Diagnostic and monitoring methods for cancer

ABSTRACT

The invention is directed to diagnostic and monitoring methods (assays) for cancer and kits that may be used in such methods. More particularly, an aspect of the invention relates to the use of activated Stat5 for diagnosing and monitoring breast cancer and predicting the effectiveness of cancer treatment. The invention also relates to the use of screening assays for discovering compounds that effect levels of activated Stat5.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention generally relates to diagnostic and monitoringmethods and assays for cancer and kits that may be used in such methods.More particularly, the application relates to the use of activated Stat5for diagnosing and monitoring cancer and predicting the prognosis of(breast) cancer patients and the outcome of cancer therapies, especiallybreast cancer. The invention also relates to screening assays fordiscovering compounds that affect levels of activated Stat5.

[0003] 2. Related Art

[0004] One of the most pressing health issues today is diagnosing,monitoring and treating cancer and particularly breast cancer. Breastcancer is the leading form of cancer in women, and the second leadingcause after lung cancer of cancer death among this population in theUnited States. In the industrialized world, about one woman in everynine can expect to develop breast cancer in her lifetime. In the UnitedStates, the annual incidence breast cancer is about 180,000 new casesand approximately 48,000 deaths each year (Parkin 1998; Apantaku 2000).Approximately two million women living in the United States alone havebeen diagnosed with breast cancer at some point in their lives. Breastcancer also occurs among men, though far more rarely (approximately1,600 new cases diagnosed in the U.S. 1998). Treatment for male breastcancer is guided by our understanding of the disease in women.

[0005] Despite ongoing improvements in understanding the disease, breastcancer has remained to a large extent resistant to medical intervention.Most clinical initiatives are focused on early diagnosis, followed byconventional forms of intervention, particularly surgery, radiation,hormone suppression, and chemotherapy. Such interventions are of limitedsuccess, particularly in patients where the tumor has undergonemetastasis. In patients with breast cancer without detectable lymph nodemetastases, socalled node negative breast cancer, the risk of death frombreast cancer recurrence within 10 years is also high, approximately 30%(McGuire, Tandon et al. 1992). There is a pressing need to improve thearsenal of diagnostic tools and methods available to provide moreprecise and more effective information that will allow successfultreatment in the least invasive way possible. Specifically, markers thatcan identify patients with very low risk of disease recurrence and deathafter initial surgery would reduce the extent of overtreatment withexpensive and potentially toxic supplementary regimes. The inventionmeets that need by providing new methods and markers for monitoringbreast cancer.

[0006] Breast Cancer

[0007] Development of cancer is a multistep process of geneticalterations that transform normal cells into highly malignantderivatives (Kinzler and Vogelstein 1996; Lengauer, Kinzler et al.1998). Tumors within the breast may arise from any of its componenttissues (e.g. connective tissue and epithelial structures). However, itis the epithelial tissue compartment that gives rise to most commonmalignant breast neoplasms.

[0008] A number of risk factors for carcinoma of the breast have beenidentified. These include: geographic influences, geneticpredisposition, increasing age, length of reproductive life, parity, ageat birth of first child, obesity, exogenous estrogens, fibrocysticchanges with a typical epithelial hyperplasia and carcinoma of thecontralateral breast or endometrium (Cole 1980; Stoll 1998). The chiefforms of carcinoma of the breast are classified as infiltrating ornoninfiltrating arising in the ducts. These include intraductalcarcinoma, comedocarcinoma, simple or usual type (including scirrhouscarcinoma), medullary carcinoma, colloid carcinoma, Paget's disease ofthe breast and tubular carcinoma. Infiltrating and noninfiltratingcarcinomas also arise in the lobules and are referred to as in situlobular carcinoma and infiltrating lobular carcinoma (Simmons andOsborne 1999; Styblo and Wood 1999).

[0009] Among the large group of breast cancer patients with localizedtumors and without detectable metastases to nearby lymph nodes, manywill be cured by surgery because the tumors have not spread tosurrounding tissues and lymph nodes. However, others have occultmicrometastatic disease and could benefit from supplementary radiationor adjuvant anti-hormone therapy or chemotherapy. There is a need fordiagnostic markers to discriminate between tumors with low risk formicrometastatic spread and those with higher risk. Tumor markers thatsignify low risk of micrometastatic disease may directly affect thetherapeutic decision of whether to use supplementary radiation oradjuvant hormone or chemotherapy. Furthermore, such tumor markers mayalso affect the surgeon's recommendation of whether to choose breastconserving surgery or mastectomy.

[0010] The molecular basis of cancer is still being determined.Underlying genome instability facilitates progressive accumulation ofgrowth-promoting traits in premalignant cells under selective pressurefrom various growth barriers (Cahill et al 1999). Growth-promotingcharacteristics of cancer include self-sufficiency in growth signals,insensitivity to anti-growth signals, evasion of apoptosis, limitlessreplicative potential, sustained angiogenesis, tissue invasion andmetastasis (Hanahan and Weinberg 2000). Associated with this stepwiseprogression of tumor cells toward increasing malignancy is a gradualloss of tissue-specific cell differentiation.

[0011] Loss of tumor cell differentiation appears to be particularlyprominent at the transition from localized, surgically curable cancer tometastatic disease (Hart and Easty 1991; Freije, MacDonald et al. 1998;Rivadeneira, Simmons et al. 2000). This transition also is the singlemost critical determinant of prognosis for patients with solid tumors(McGuire 1991; Tubiana 1999). Assessment of the activity oftranscriptional regulators that maintain cell and tissue-specificdifferentiation in primary tumors may therefore be useful for predictingthe risk of occult micrometastases and tumor recurrence. Suchinformative tumor markers may directly influence treatment decisions byeither providing prognostic distinction between low- and high-riskmalignancies, or by predicting tumor response to specific adjuvanttherapies or tumor response to specific modes of surgery (breastconservation surgery vs. mastetomy).

[0012] In breast cancer, receptors for estrogen and progesterone arerelated to the state of mammary epithelial cell differentiation and haveprognostic value for disease outcome in certain cases. Estrogen andprogesterone receptor (ER/PR) status is particularly useful as apredictive marker of positive response to adjuvant anti-estrogen therapyin node-positive breast cancer. However, the ER/PR status is notclinically useful to predict prognosis in node-negative cancer(Fitzgibbons, Page et al. 2000). This may be due to the high proportionof ER/PR positive, localized tumors. There is a need to identifylow-risk breast cancer patients who may be spared from costly andpotentially toxic adjuvant antiestrogen treatment or chemotherapy. Thereis also a need to identify low-risk breast cancer patients who maybenefit from less invasive procedures such as breast conserving surgery,or lumpectomy, with or without post-surgical radiation therapy, insteadof mastectomy. The benefits of less extensive and less invasivetherapeutic regimes to patients with good prognosis may includeavoidance of side-effects, improved mental and physical health, improvedquality of life, and lower financial burden. The benefits to society areparticularly the cost-saving aspects of avoiding unneccessaryovertreatment. One means of accomplishing this is to obtain betterprognostic markers for node-negative, as well as other types of breastcancer. These needs are met by the invention.

[0013] Diagnosis of Breast Cancer

[0014] The definitive diagnosis of all types of breast disease is basedon histologic evaluation of tissue samples using the light microscope.The histologic criteria used to define most breast lesions are historicbut nonetheless quite reproducible for identifying fully invasive breastcancers.

[0015] Improved detection and screening routines, and the developmentand increasing utilization of fine needle aspirates (FNAs) and coreneedle biopsies for obtaining tissue samples have been major advances inboth detection and diagnosis. Stereotactic image guidance of needlebiopsies has tremendously improved our ability to sample suspiciouslesions, particularly non-palpable masses, as small as a few millimetersin diameter nearly anywhere in the breast. This has dramaticallyincreased the detection of small, more treatable breast cancers anddecreased unnecessary surgery in an enormous number of patients withinsignificant benign disease. Recent accomplishments include theidentification of a small number of tissue-based biomarkers that arehelpful in predicting clinical outcome and response to therapy (e.g.,S-phase fraction, estrogen and progesterone receptors, c-erbB-2) and thediscovery of genes (BRCA-1 and BRCA-2) associated with familial risk forbreast cancers (Dahiya and Deng 1998; Fitzgibbons, Page et al. 2000).

[0016] However, diagnosing breast cancer still requires some type ofbiopsy procedure. In addition, current diagnostic and prognostic methodscannot absolutely distinguish breast cancers that are treatable bysurgery alone from those that are likely to recur or have already spreadthrough micrometastases. As a result, at least 50 percent of breastcancer patients with node negative disease are treated with some form ofadjuvant therapy. Moreover, available methods are inadequate forpredicting the response of breast cancers to specific types of adjuvanttherapies.

[0017] Treatment decisions for individual breast cancer patients arefrequently based on the number of axillary lymph nodes involved withdisease, estrogen receptor and progesterone receptor status, size of theprimary tumor, and stage of disease at diagnosis (Tandon, Clark et al.1989). However, even with this variety of factors, it is currently notpossible to predict accurately the course of disease for all breastcancer patients. There is clearly a need to identify new markers inorder to separate patients with good prognosis, who might need nosupplementary therapy beyond surgical removal of the malignant breasttumor, from those whose cancer is more likely to recur and who mightbenefit from additional and more exhaustive treatment forms.

[0018] Despite extensive efforts over several decades, only a limitednumber of immunohistochemical breast tumor markers have been identified.Among immunohistochemical markers, hormone receptor status remains theonly to have gained standard clinical use for evaluating node-negativebreast tumors (Fitzgibbons, Page et al. 2000). With improving methodsfor screening and detection of early breast cancer the proportion ofnode-negative cases is expected to continue to rise (Elledge and McGuire1993). Parameters that have been established to be important for theprognosis of patients with breast malignancies in general and that areused by clinicians include: size of primary tumor, stage of disease atdiagnosis, number of axillary lymph nodes involved with disease, andhormonal receptor status (ER/PR) (Fitzgibbons, Page et al. 2000).Abnormal status of ErbB-2 or p53, as well as other histological andgenetic markers, also are associated with poor prognosis especially innode-positive tumors (Slamon, Clark et al. 1987; Fresno, Molina et al.1997; Pharoah, Day et al. 1999).

[0019] In this regard, U.S. Pat. No. 5,599,681 has suggested the use ofan antibody that specifically binds to a reversible phosphorylation siteof the c-erbB2 oncoprotein in its active form to screen for themetastatic potential of tumors in patients with node-negative breastcancer. Nowhere, however, was it suggested that screening for activatedStat5 could be used to predict the metastatic potential of breastcancer.

[0020] There remain deficiencies in the art with respect to theidentification of markers linked with the progression of breast cancer,the development of diagnostic methods to monitor disease progression andthe development of therapeutic methods and compositions; to treat breastdiseases and cancers. The identification of markers which aredifferentially expressed or activated in breast cancer would be ofconsiderable importance in the development of a rapid, inexpensivemethod to improve diagnosing of breast cancer and to predict tumorbehavior with respect to patient prognosis and responsiveness toindividual therapeutic options. The identified marker(s) would also beuseful as a target of therapeutic compositions, of in screening assaysfor therapeutic compounds.

[0021] The diagnostic and monitoring methods of the invention meet manyneeds in this area.

[0022] Therapeutic Regimes for Treating Breast Cancer

[0023] Treatment of breast cancer is multifaceted and complex. Thechoice of therapeutic approach is guided by a series of criteria basedon a limited set of tumor characteristics. Nearly all patients withbreast cancer will have some type of surgery. This may be supplementedby local therapy with radiation, or by systemic therapy includinghormone suppression or chemotherapy. To kill cancer cells that may havespread beyond the breast and nearby tissues, physicians employ oral orintravenous systemic therapy. Examples of systemic treatments for breastcancer are chemotherapy and antiestrogen therapy. Systemic therapy givento patients after surgery is often referred to as adjuvant therapy. Thegoal of adjuvant therapy is to kill hidden cancer cells. Even in theearly stages of the disease cancer cells can break away from the primarybreast tumor and spread through the bloodstream. These cells usuallycause no detectable symptoms and usually do not show up on an x-ray andcannot be felt during a physical examination. But they can establish newtumors in other locations in the body. Furthermore, oncologistssometimes give patients neo-adjuvant therapy—that is, systemic therapybefore surgery, typically to shrink the tumor.

[0024] The following summarizes the main principles of treatment ofbreast cancer according to current guidelines endorsed by the U.S.National Cancer Consortium Network and the American Cancer Society(1999). The text below maintains an emphasis on treatment ofnode-negative breast cancer, as it relates to the present invention.

[0025] Breast conserving surgery—“Lumpectomy” removes only the breastlump and the surrounding area, or margin, of normal tissue. If cancercells are present at the margin (the edge of the excisional biopsy orlumpectomy specimen), a re-excision can usually be done to remove theremaining cancer. In most cases, lumpectomy is combined with 6 to 7weeks of supplementary radiation therapy following surgery. Thiscombination of lumpectomy and radiation is often referred to as “breastconserving therapy”.

[0026] Mastectomy—In a “simple (total) mastectomy” procedure surgeonsremove the entire breast but do not remove any lymph nodes from underthe arm, or muscle tissue from beneath the breast. In a “modifiedradical mastectomy”, surgeons remove the entire breast and some of theaxillary (underarm) lymph nodes. Modified radical mastectomy is the mostcommon surgery for patients with breast cancer in whom doctors removethe whole breast. “Radical mastectomy” removes not only the entirebreast, but axillary lymph nodes and the chest wall muscles under thebreast as well. The less extensive modified radical mastectomy hasproved as effective as radical mastectomy, which is nowadays rarelyperformed due to disfiguration and frequent side-effects.

[0027] Lymph node surgery—Regardless of whether a breast cancer patienthas a mastectomy, or a lumpectomy for invasive cancer, the physiciansneed to determine whether the cancer has spread. The regional lymphnodes in the underarm drain lymph from the breast, and are typically thefirst sites of spread. Furthermore, lymph node involvement increases thelikelihood that cancer cells have spread through the blood-stream toother parts of the body.

[0028] While lymph node surgery itself does not improve the chance for acure, this is the only way to accurately determine if the cancer hasspread to the lymph nodes. This usually means removing some or all ofthe lymph nodes in the armpit. Typically 10 to 20 lymph nodes in thearmpit are examined by an operation called “axillary lymph nodedissection”. Although axillary lymph node dissection is a safe procedurewith low rates of serious side effects, efforts are ongoing to developnew ways of detecting the spread of cancer to lymph nodes that are lessinvasive and do not involve a full lymph node dissection. Suchalternative methods include the “sentinel lymph node biopsy” (Orr, Hoehnet al. 1999; Sugg, Ferguson et al. 2000), and new detection methods forbreast cancer cells in bone marrow and blood (Berois, Varangot et al.2000; Braun, Pantel et al. 2000; Fetsch, Cowan et al. 2000; Ikeda,Miyoshi et al. 2000; Kraeft, Sutherland et al. 2000; Zhong, Kaul et al.2000). It is possible that these newer methods in the future may replacelymph node dissection as a means of determining micrometastatic spreadof cancer.

[0029] Sentinel lymph node biopsy—In the sentinel lymph node biopsyprocedure the surgeon finds and removes the ‘sentinel node’—the firstlymph node into which a tumor drains, and therefore the one most likelyto contain cancer cells. Many doctors recommend it for most women withbreast cancer, but others still consider it investigational. In asentinel lymph node biopsy the surgeon injects a radioactive substanceand/or a blue dye into the area around the tumor. Lymphatic vesselscarry these materials into the sentinel node. The doctor can either seethe blue dye or detect the radioactivity with a geiger counter, and thencuts out the node for examination. If the sentinel node contains cancer,the surgeon will have to perform an axillary dissection—removal of morelymph nodes in the axilla (armpit). If the sentinel node is cancer-free,the patient and her physicians may consider avoiding more lymph nodesurgery and its potential side effects. Although the sentinel nodeprocedure is relatively new and its long-term effectiveness is uncertain(Orr, Hoehn et al. 1999; Sugg, Ferguson et al. 2000), it may turn out tobe equally as effective in determining lymph node spread as full lymphnode dissection.

[0030] Detection of disseminated cancer cells in blood and bonemarrow—Recent methods for detecting metastatic breast cancer cells inblood (Berois, Varangot et al. 2000; Fetsch, Cowan et al. 2000; Kraeft,Sutherland et al 2000) or in bone marrow (Braun, Pantel et al. 2000;Ikeda, Miyoshi et al. 2000; Zhong, Kaul et al. 2000) are typically basedon the detection of cytokeratin markers characteristic to breast cancercells by immunological methods or by gene-based testing. These newmethods may also lead to an alternative approach to lymph nodedissection for determining whether a breast cancer has spread beyond thelocal tumor area.

[0031] Radiation therapy—Radiation is used to destroy cancer cells leftbehind in the breast, chest wall, or lymph nodes after surgery.Radiation treatments usually take place 5 days a week over a period of 6to 8 weeks. Side effects most likely to occur include swelling andheaviness in the breast, sunburn-like skin changes in the treated area,and fatigue. Changes to the breast tissue and skin usually go away in 6to 12 months. In some women, the breast becomes smaller and firmer afterradiation therapy. Radiation therapy of axillary (armpit area) lymphnodes can also cause lymphedema. Although generally safe, it is evidentthat radiation therapy comes at a considerable expense and withpotentially serious side-effects. Radiation therapy also involves amajor risk for abnormal fetal development, and cannot be used to treatpregnant women with breast cancer.

[0032] New tumor markers that signify good prognosis may reduce the needfor supplementary radiation therapy.

[0033] Chemotherapy—Patients receive this treatment of anti-cancer drugsintravenously (injected into a vein) or by mouth. Either way, the drugstravel in the bloodstream and move throughout the entire body. Doctorswho prescribe these drugs (medical oncologists) generally use acombination of medicines proven more effective than a single drug. Forwomen with node-negative breast cancer the most frequently usedchemotherapy options are CMF (cyclophosphamide, methotrexate, andfluorouracil), CAF (cyclophosphamide, doxorubicin), and AC (doxorubicin(Adriamycin) and cyclophosphamide) (1999). Doctors give chemotherapy incycles, with each period of treatment followed by a recovery period. Thetotal course of chemotherapy usually lasts 3 to 6 months depending onthe combinations used. This is significant both in terms of cost andreduced well-being. The side effects of chemotherapy are many andpotentially severe, and depend on the type of drugs used, the amounttaken, and the length of treatment. Doxorubicin and epirubicin may causeheart damage, although doctors limit the dose and perform periodic teststo check heart function in order to prevent this side effect. Other sideeffects include loss of appetite, nausea and vomiting, mouth sores, hairloss, and changes in the menstrual cycle. Because chemotherapy candamage the blood-producing cells of the bone marrow, a drop in whiteblood cells can raise a patient's risk of infection, a shortage of bloodplatelets can cause bleeding or bruising after minor cuts or injuries;and a decline in red blood cells can lead to fatigue due to anemia.

[0034] New tumor markers that identify patients with excellent prognosismay eliminate the need for adjuvant chemotherapy among these patients.

[0035] Hormone therapy—Estrogen, a female sex hormone produced by theovaries, promotes growth of some breast cancers. Doctors use severalapproaches to block the effect of estrogen or to lower estrogen levels.The most commonly used antiestrogen drug is tamoxifen, taken daily inpill form, usually for 5 years. Studies show that tamoxifen can reducethe chances of breast cancer coming back after surgery if the breastcancer cells contain receptors for estrogen or progesterone. Tamoxifenmay be used to treat metastatic breast cancer, but also a significantnumber of patients with node-negative cancer receive tamoxifentreatment.

[0036] Adjuvant Herceptin therapy—A new form of adjuvant breast cancertreatment involves the use of Herceptin, a drug that antagonizesactivity of the Her2/neu oncogene reecently introduced for selectpatients with node-positive breast cancer (Stebbing, Copson et al.2000). Herceptin therapy will not be discussed in more detail here.

[0037] Therapeutic considerations in node-negative breastcancer—Decisions about types of surgery (breast conserving lumpectomy,radical mastectomy), radiation therapy, adjuvant chemotherapy orhormonal therapy are currently based on the status of axillary lymphnodes, the size of the malignant tumor and its histologic type(appearance under a microscope), and hormone receptor status. Forexample, if regional lymph nodes are negative (do not contain any cancercells) and the tumor measures half a centimeter or smaller, the patientneeds no adjuvant (post-surgery) therapy. In current practice, asubstantial number of patients with node-negative breast cancer withlarger tumors receive adjuvant therapies with questionable benefit interms of relatively limited improvement in prognosis considering theassociated increased morbidity and serious side-effects (McGuire, Tandonet al. 1992). These adjuvant therapies also come at high cost asdescribed above. Furthermore, the choice of the less invasive breastconserving surgery (lumpectomy) is generally preferred by doctors andpatients over mastectomy, but more specific guidelines and betterprognostic tumor markers are needed to guide this selection. There istherefore a strong need for new markers to identify breast cancerpatients with low risk for disease recurrence and death.

[0038] Markers for low-risk cancer and patient follow-up—Betterprognostic tumor markers may also have the benefit of reducing thefrequency of follow-up visits among patients with low-risk cancer. Tumormarkers identifying low-risk breast cancer patients may also allowreduced frequency and lighten the extensive requirements for patientfollow-up. While this is primarily a cost issue, it also positivelyimpacts the patient's quality of life. Routine surveillance andfollow-up for all patients who have had invasive breast cancer curentlyincludes the following: a history and physical exam every 4-6 months for2 years, then every 6 months for 3 years, and then, once every year(1999). Women who have had a lumpectomy and radiation (breastconservation therapy) should undergo mammography of the treated breastat 6 months after radiation therapy, and then mammography of bothbreasts on an annual basis. Women who have had a mastectomy should get amammogram of the remaining breast annually after the surgery. Becausetamoxifen increases a postmenopausal woman's risk of developing cancerof the endometrium (lining of the upper part of the uterus),postmenopausal patients taking this drug also should have an annualpelvic exam. Markers indicating low-risk for tumor recurrence thereformay benefit both patients and society by reduced costs associated withfewer and less extensive follow-up examinations.

[0039] Monitoring of recurrent breast cancer—Work-up for a suspectedrecurrence of breast cancer includes a biopsy to confirm the firstrecurrence whenever possible. A recurrence may be local, meaning thatcancer has returned to the breast, underarm lymph nodes, or nearbytissues, or systemic, which means that cancer has spread to distantorgans. There exist a series of guidelines to treat locally recurringbreast cancer. The current recommendations for treatment of the locallyrecurring tumor depend in large part on what mode of treatment was usedfor the original tumor (1999). New markers that predict the biologicalbehavior of breast cancer may affect the choice of follow-up therapy,depending on whether the recurrent tumor is deemed low or high risk. Forinstance, local recurrence of a tumor positive for a marker indicatinglow risk of distant spread may allow the use of less intensivetherapeutic approaches than if the tumor is negative for this samemarker. For example, reexcision and possibly local radiation may sufficeinstead of radical mastectecomy with or without adjuvant chemotherapy oranti-hormone therapy.

[0040] Stat5

[0041] The Signal Transducer and Activator of Transcription (STAT)family of transcription factors provide a signaling link between cellsurface hormone and cytokine receptors and specific response elements inthe promoters of selective genes. Seven mammalian STAT genes have beenidentified. The Stat5 transcription factor is involved in regulation ofcell growth, differentiation, and cell survival (Wakao, Gouilleux et al.1994). It exists as two highly homologous isoforms, Stat5a and 5b, whichhave more than 95% amino acid homology and are encoded by separate genes(Liu, Robinson et al. 1995; Grimley, Dong et al. 1999). Stat5 isrequired for normal mammary epithelial cell development anddifferentiation (Liu, Robinson et al. 1997; Udy, Towers et al. 1997;Moriggl, Topham et al. 1999).

[0042] Stat5 polypeptides typically are cytoplasmic and quiescent underhomeostatic conditions. Their activation results from phosphorylation ofthe highly conserved C-terminal tyrosine at Tyr694 in Stat5a or thecorresponding Tyr699 in Stat5b by certain intracellular tyrosinekinases. This phosphorylation permits dimer pair formation which isneeded for Stat5 to bind to DNA.

[0043] This initial phosphotyrosyl “on-switch” is a generic Stat feature(Darnell 1997; Darnell 1998) and is triggered when cells with cognatereceptors are exposed to a variety of stimuli including cytokines,immune complexes, microbiologic agents or non-peptidyl compounds.Although the spectrum of agonists thus is heterogeneous, the bulkimplicated in triggering Stat5 activation belong to the class I andclass II cytokine superfamilies. (See Table 4 of (Grimley, Dong et al.1999). These cytokines utilize receptors lacking a catalytic domain(Liu, Gaffen et al. 1998), so that the Stat activation is most oftendependent upon an auxiliary protein kinase (Leonard and O'Shea 1998).

[0044] The Janus tyrosine kinases (Jaks) form biochemically stableassociations with class I and class II cytokine receptors. Anon-covalent linkage facilitates Jak phosphorylations during receptorligation and increases the odds of interactions between Jaks and Stat5recruited to receptor-Jak complexes (Leonard and O'Shea 1998). Thiscritical and conserved mutual relationship has engendered the scientificvernacular of “Jak-Stat pathway” (Liu, Gaffen et al. 1998). However,Jaks are not the sole means of Stat activation.

[0045] Stat5a and Stat5b can also be tyrosine phosphorylated by a numberof cytokines commonly designated as “growth factors” which bind toreceptor tyrosine kinases (RTKs). The RTKs possess intrinsic catalyticproperties, and may trigger Stat5 signals absent a direct linkage to theJak enzyme system (Chen, Sadowski et al. 1997). In addition, Stat5tyrosine phosphorylation might be effected by cytosolic protein kinasesin the Src or Tec families. As “nonreceptor tyrosine kinases” (NTKs),the latter enzymes can function without extrinsic stimulation due toreceptor ligation. The Src-family kinase Lck has been implicated inStat5 phosphorylation during T cell proliferation (Welte, Leitenberg etal. 1999) and constitutively active NTKs, RTKs or analogous oncoproteinsmay be particularly significant in maintaining a constitutivephosphorylation of Stat5 in autonomously proliferating neoplastic cells(For example, See (Lacronique, Boureux et al. 1997; Wellbrock,Geissinger et al. 1998)).

[0046] In addition to the initial activation switch of Stat5, whichinvolves phosphorylation of a tyrosine residue within a conservedC-terminal segment and causes dimerization of Stat5 molecules(Gouilleux, Wakao et al. 1994), a second coordinated activation event isrequired for functional activation. This involves translocation ofdimerized Stat5 from the cytoplasm into the cell nucleus, which permitsStat5 to come in proximity of and bind to gene regulatory promoterelements, and thus regulate transcription of specific genes (Gouilleux,Wakao et al. 1994; Kazansky, Kabotyanski et al. 1999). Because Stat5 notonly requires phosphorylation of a specific tyrosine residue, but alsoneeds to translocate into the cell nucleus in order to function as anactive DNA-binding transcription factor, amounts of tyrosinephosphorylated Stat5 located within the cell nucleus will reflect thelevels of activated Stat5 more accurately than overall cellular levelsof tyrosine phosphorylated Stat5. For instance, tyrosine phosphorylationof Stat5a by the Src tyrosine kinase has been shown not to beaccompanied by nuclear translocation (Kazansky, Kabotyanski et al.1999), illustrating that quantitation of tyrosine phosphorylation statusalone without assessing nuclear localization is not sufficient foraccurate determination of levels of activated Stat5. Correspondingly,Stat transcription factors may become dephosphorylated within the cellnucleus and loose the ability to bind to DNA (Haspel and Darnell 1999),making assays that detect nuclear Stat5 protein levels alone also notsufficient for accurate determination of levels of activated Stat5. Inthe present description, the term ‘levels of activated Stat5’ refers tolevels of tyrosine phosphorylated Stat5 within the cell nucleus.

[0047] Antibodies that bind exclusively to tyrosine phosphorylated Stat5can be used to detect activated Stat5 in the nuclei of cells byimmunocytochemistry or immunohistochemistry, provided that proper stepsare taken to achieve antigen retrieval of this cryptic antigenic site.This antigenic site is cryptic, or unavailable, unless thephosphorylated tyrosine bound to the SH2 domain of the partner moleculein the dimer is dissociated by specific treatment.

[0048] Detection of active, tyrosine phosphorylated Stat5 byimmunohistochemistry in tissue sections has been reported (Jones, Welteet al. 1999). Stat5 activation in normal mouse mammary gland tissue inresponse to Erb-B4 activation was studied. However, human breast tissueor human breast cancer samples were not examined. In further contrast toJones, the current invention may use a simple one-step antigen-retrievalmethod for determining levels of activated Stat5.

[0049] The extent to which Stat5 promotes cell proliferation or inhibitsgrowth by inducing cell differentiation in various tissues, includingmammary gland, is unresolved. The possibility that Stat5 activationstatus is of prognostic value for breast cancer was not obvious prior tothe inventors' discovery, because a priori, it had been argued thatStat5 activation may promote mammary tumor formation instead of beingassociated with reduced risk of invasion and metastasis.

[0050] It was specifically suggested that a general anti-apoptoticeffect of Stat5 might contribute to mammary tumor progression in rodents(Humphreys and Hennighausen 2000). This notion was supported by theobservation that in mice lacking the Stat5a gene (Stat5a−/− mice) butoverexpressing the oncogenic TGF-alpha transgene, the rate of mammarytumor formation was reduced relative to that observed in Stat5a+/+ mice(Humphreys and Hennighausen 1999)). This suggested that the Stat5atranscription factor promotes mammary tumor formation. Likewise, apositive role for Stat5 in mammary carcinogenesis indirectly has beenindicated by the reduced mammary tumor formation in mice lacking thegene for prolactin, a major activator of Stat5 in mammary epithelialcells (Vomachka, Pratt et al. 2000), as well as the observation thatcirculating prolactin levels correlated with increased risk of breastcancer in post-menopausal women (Hankinson, Willett et al. 1999).Furthermore, the notion of a tumorigenic role of Stat5 in the mammarygland (Humphreys and Hennighausen 1999; Humphreys and Hennighausen 2000)would be consistent with the prevailing view of a tumor-promoting roleof Stat5 in hematopoietic cancer (lymphomas, leukemias) (Wellbrock,Geissinger et al. 1998; Bromberg and Darnell 2000).

[0051] Alternatively, it could be argued that Stat5 activation maysuppress breast tumor formation by acting as a growth-inhibitorydifferentiation factor for mammary epithelial cells. Likewise, Stat5regulates normal differentiation of ovaries and prostate (Teglund, McKayet al. 1998; Moriggl, Topham et al. 1999; Nevalainen, Ahonen et al.2000). However, there is currently no direct evidence availabledemonstrating a role of Stat5 as a tumor suppressor in either breast orother tissues. Therefore, the present invention and description ofactivated Stat5 as a marker of good prognosis in node-negative humanbreast cancer was unexpected based on the published literature andprevailing views within the scientific field. As such, the role of Stat5in human breast cancer development and progression had not beenestablished, and its use as a marker of biologic behavior of humanbreast tumors had not been reported.

SUMMARY OF THE INVENTION

[0052] It has been discovered by the inventors that activated Stat5within human primary breast tumors correlates with reduced risk of deathfrom breast cancer and reduced risk of metastatic disease. Thiscorrelation was particularly strong for node-negative breast cancer.Such a correlation was not known or even suspected prior to theinventors' discovery. Therefore, nuclear, activated Stat5 is a new andnovel prognostic marker of breast cancer.

[0053] The presence of nuclear, activated Stat5 in human breast cancerindicates a higher degree of differentiation of the tumor and is alsoassociated with an increased survival rate within this patientpopulation. Activated Stat5 levels in the primary tumor of node negativebreast cancer patients is a strong positive prognostic factorindependent of other known prognostic markers, as evidenced bymultivariate Cox regression analysis.

[0054] Levels of activated, nuclear Stat5 may be analyzed withantibodies or other binding probes. Thus, the analysis of activatedStat5 adds a new level of information to current breast cancer markersand is a reliable prognostic molecular/biochemical marker of cancer insamples from untreated breast cancer patients. Additionally, monitoringlevels of activated Stat5 should be predictive of the outcome ofStat5-targeted therapeutic strategies.

[0055] The invention involves the use of activated Stat5 as a tumormarker that predicts the risk/prognosis and biological behavior ofbreast cancer. The inventors describe a straight-forward method todetermine Stat5 activation status in histological tissue sections oftumors by immunohistochemistry. In this regard, analysis of nuclear,activated Stat5 levels, by univariate Cox regression analysis can alsobe used as a predictive measure of a positive outcome of antiestrogentreatment. Furthermore, it may also be predictive of the success ofother treatments where alterations in Stat5 activation levels areinvolved, as well as predictive of success of breast conserving surgeryand radical mastectomy.

[0056] The invention is first directed to a diagnostic or monitoringmethod comprising: a) obtaining a sample of tissue from an individual inneed of diagnosis or monitoring for cancer; b) detecting levels ofactivated Stat5 antigen in said sample; c) scoring said sample foractivated Stat5 levels; and d) comparing said scoring to that obtainedfrom a control tissue sample to determine the prognosis associated withsaid cancer. Cancers that may be diagnosed or monitored include but arenot limited to breast cancer, ovarian cancer, endometrial cancer,thyroid cancer, prostate cancer, colorectal cancer, hematopoieticcancer, and skin cancer.

[0057] The invention is further directed to a diagnostic or monitoringmethod comprising: a) obtaining a sample of breast tissue from anindividual in need of diagnosis or monitoring for breast cancer; b)detecting levels of activated Stat5 antigen in said sample; c) scoringsaid sample for activated Stat5 levels; and d) comparing said scoring tothat obtained from a control breast sample to determine the prognosisassociated with said breast cancer. Preferably, the cancer is a nodenegative breast cancer.

[0058] The invention is also directed to a diagnostic or monitoringmethod comprising: a) obtaining a sample from an individual in need ofdiagnosis or monitoring for breast cancer; b) contacting said samplewith an antibody or binding probe that detects activated Stat5; c)detecting or measuring the level of activated Stat5; and d) comparingthe level of activated Stat5 to that obtained from a control breastsample.

[0059] In all aspects of the invention a preferable embodiment involvescontacting the sample of interest with an antibody totyrosine-phosphorylated Stat5. Preferably the detecting is done onhistological or tissue sections or cytological preparations byimmunohistochemistry or immunocytochemistry. Additionally, detecting ofactivated Stat5 in the methods of the invention may be done byimmunoblotting or by Fluorescence-Activated Cell Sorting (FACS).

[0060] The invention is also directed to a method for screeningcompounds comprising: a) obtaining compounds to be screened for use inbreast cancer therapy; b) contacting a cell or tissue sample with saidcompound; and c) determining the effect of said compound on the level ofStat5 activation in said cell or tissue sample relative to a controlsample. Preferably the cell or tissue sample is cells or tissue from abreast cancer. Additionally, in the method of the invention the effectof said compound may be determined by the binding of an antibody toactivated Stat5 to said sample relative to control cells or tissue. Alsopreferably in the method said activated Stat5 is tyrosine-phosphorylatedStat5 found in the cell nucleus.

[0061] The invention is further directed to a method for screeningcompounds comprising: a) obtaining compounds to be screened for alteringStat5 activation levels, b) contacting a cell or tissue of interest withsaid compounds, c) determining the effect of said compound on the levelof activated Stat5 in said cell or tissue sample relative to a controlsample.

[0062] The invention is also directed to a method for screeningcompounds comprising: a) obtaining compounds to be screened for use incancer therapy; b) contacting a cell or tissue sample with saidcompound; and c) determining the effect of said compound on the level ofStat5 activation in said cell or tissue sample. Preferably the cell ortissue sample is from a human cancer. The effect of said compound may bedetermined by the binding of an antibody to activated Stat5 to saidsample relative to control cells or tissue. Preferably said activatedStat5 is tyrosine-phosphorylated Stat5 found in the cell nucleus.

[0063] The invention is also directed to a method for screeningcompounds comprising: a) obtaining compounds to be screened for theirability to positively or negatively affect Stat5 activation; b)contacting a relevant cell or tissue sample with said compound; and c)determining the effect of said compound on the level of Stat5 activationin said cell or tissue sample. Preferably the effect of said compoundmay be determined by the binding of an antibody to activated Stat5 tosaid sample relative to control cells or tissue. Also preferably saidactivated Stat5 is tyrosine-phosphorylated Stat5 found in the cellnucleus.

[0064] The invention is also directed to a method for determining theeffect of antiestrogen treatment comprising: a) obtaining a cell ortissue sample from an individual in need of antiestrogen treatment, b)measuring the levels of activated Stat5 in said cell or tissue sample;and c) comparing said levels to that of a control breast cancer sampleto predict the responsiveness to antiestrogen treatment.

[0065] The invention is also directed to a method for determining theefficacy of breast conserving surgery (lumpectomy) for treatment ofnode-negative breast cancer comprising: a) obtaining a cell or tissuesample from an individual in need of breast conserving surgery, b)measuring the levels of activated Stat5 in said cell or tissue sample;and c) comparing said levels to that of a control breast cancer sampleto predict the responsiveness of said breast cancer to breast conservingsurgery.

[0066] The invention is further directed to a kit for determining thelevel of activated Stat5 in a mammalian biological sample, wherein saidactivated Stat5 is an indicator of the prognosis of breast cancer, saidkit comprising: a) an antibody or binding probe to activated Stat5, b) areagent useful for detecting the extent of interaction between saidantibody or binding probe and activated Stat5; c) a reagent or solutionuseful for antigen retrieval; and c) positive and/or negative controlsamples. The kit of invention may include a monoclonal or polyclonalantibody as the antibody. This antibody may be directly linked to anindicator reagent, wherein said indicator reagent is selected from thegroup consisting of fluorescent, colorimetric, immunoperoxidase andisotopic reagents. Alternatively, the kit may further include a secondindicator antibody linked to an indicator reagent, wherein saidindicator reagent is selected from the group consisting of fluorescent,calorimetric, immunoperoxidase and isotopic reagents.

[0067] The invention is further directed to a method for diagnosing apathological condition or a susceptibility to a pathological conditioncomprising: a) obtaining a sample from an individual in need ofdiagnosis for a pathological condition related to activity of Stat5, b)determining the amount or presence of activated Stat5 in said sample;and c) diagnosing said pathological condition or a susceptibility tosaid pathological condition based on the presence or amount of activatedStat5 relative to a control sample.

[0068] Any of the methods of the claimed invention may use eitherunivariate or multivariate Cox regression analysis or Kaplan-Meyersurvival analysis with log-rank statistics for analyzing the obtainedresults or may analyze a sample in a tissue section, isolated cell, orisolated nuclei (smears, cytological sample or flow cytometry.) Themethods of the invention may further comprise analyzing the levels ofactivated Stat5 in conjunction with additional breast cancer markers.

[0069] The invention is further directed to a diagnostic or monitoringmethod comprising: a) obtaining a sample of breast tissue from anindividual in need of diagnosis or monitoring for breast cancer; b)treating said sample in a microwave oven or by other forms of heat basedantigen retrieval methods; c) detecting levels of activated Stat5antigen in said sample; d) scoring said samples for activated Stat5levels; and e) comparing said scoring to that obtained from a controlbreast sample to determine the prognosis associated with said breastcancer. In addition to heat based antigen retrieval methods, othermethods known in the art for antigen retrieval may also be used.

[0070] The invention is further directed to a diagnostic or monitoringmethod comprising: a) obtaining a sample of breast tissue from anindividual in need of diagnosis or monitoring for breast cancer, b)treating said sample with an antigen-retrieval buffer, c) detectinglevels of activated Stat5 antigen in said sample; d) scoring saidsamples for activated Stat5 levels; and e) comparing said scoring tothat obtained from a control breast sample to determine the prognosisassociated with said breast cancer. Preferably said antigen retrievalsolution is an aqueous buffer about pH 7 to about pH 10, such as forexample Phosphate Buffered Saline at pH 7.4. Most preferably, theantigen retrieval solution is about 1 mM Tris having about a pH 10.

[0071] The invention is further directed to a method for predictingdisease-free survival and overall survival in patients withnode-negative breast cancer comprising: a) obtaining a sample of breastcancer tissue from an individual with node-negative breast cancer; b)detecting levels of activated Stat5 antigen in breast cancer cells orbreast cancer tissue of said sample; c) scoring said samples foractivated Stat5 levels; and d) comparing said scoring to that obtainedfrom a control breast sample to determine likelihood of disease-freesurvival and overall survival associated with said breast cancer.

[0072] Any of the methods of the invention may score the analysis byusing a scale of 0 to 4, where 0 is negative (no detectable activatedStat5 in cell nuclei), and 4 is high intensity staining in the majorityof cell nuclei and wherein a score of 1 to 4 (i.e. a positive score)indicates a better prognosis for disease free and overall survival inpatients with node-negative breast cancer.

[0073] The invention is also directed to a method for predictingdisease-free survival and overall survival in patients who have notreceived adjuvant hormone or chemotherapy comprising: a) obtaining asample of breast tissue from an individual with breast cancer who hasnot received adjuvant hormone or chemotherapy, b) detecting levels ofactivated Stat5 antigen in breast cells or breast tissue of said sample,c) scoring said sample for activated Stat5 levels; and d) comparing saidscoring to that obtained from a control breast sample to determine thelikelihood of disease-free survival and overall survival associated withsaid breast cancer.

[0074] The invention is further directed to a method for treating breastcancer comprising: a) obtaining a sample of breast tissue from a patientin need of treatment of breast cancer, b) determining the level ofactivated Stat5 in said breast tissue sample, c) treating said patientwith a therapeutic regime known to improve the prognosis for breastcancer, d) repeating steps “a” and “b”, e) adjusting the therapeuticregime based on the determination of the activated Stat5 levels and f)repeating steps a-e as frequently as deemed appropriate.

[0075] The invention is further directed to a method for screening formetastatic potential of breast tumors comprising: a) obtaining a sampleof breast tissue from an individual in need of screening for metastaticpotential of a breast tumor, b) reacting an antibody to activated Stat5with tumor tissue from said patient, c) detecting the extent of bindingof said antibody to said tissue and d) correlating the extent of bindingof said antibody with its metastatic potential Preferably, the tumor isa node-negative breast cancer.

[0076] The invention is further directed to a method for screening formetastatic potential of solid tumors comprising: a) obtaining a sampleof tumor tissue from an individual in need of screening for metastaticpotential of a solid tumor; b) reacting an antibody to activated Stat5with tumor tissue from said patient; c) detecting the extent of bindingof said antibody to said tissue and d) correlating the extent of bindingof said antibody with its metastatic potential. Preferably, the tumor isa node-negative cancer arising from the ovary, large bowel (colorectalcancer), uterus (endometrial cancer), thyroid gland, prostate, or skin.

[0077] Any of the methods of the invention involving analysis of thelevels of activated Stat5 may be used in conjunction with additionalbreast cancer markers readily known to those of skill in the art.

[0078] The invention if further directed to a monoclonal antibody,wherein said antibody a) is generated against the phosphopeptide KAVDG(phospho Y)VPQIK); b) specifically recognizes tyrosine phosphorylatedisoforms of of Stat5, but not unphosphorylated isoforms; c) does notrecognize Stat5 mutants in which the Tyr694 residue has been substitutedwith phenylalanine; and d) recognizes phosphorylated Stat5 following anantigen retrieval treatment that does not use a protease.

BRIEF DESCRIPTION OF THE FIGURES

[0079]FIG. 1—Cutpoint analysis of scores quantifying levels of activatedStat5. The effect of various levels of activated Stat5 on overallsurvival of breast cancer patients is shown. Patients with primarytumors with no detectable level of activated Stat5 (Score 0) hassignificantly lower rates of overall survival than patients withdetectable levels of activated Stat5 in their tumors (Scores 1-4).However, no significant difference was noted between scores 1-4 in termsof overall survival rate. Scores 1-4 were therefore recoded into asingle categorical parameter (“Positive Stat5 activation status”),whereas score 0 was given the designation “negative Stat5 activationstatus”.

[0080] FIGS. 2A-2B—Detection of activated Stat5 by AX1 antibody.Immunocytochemistry (FIG. 2A) and immunoblotting (FIG. 2B) of activatedStat5 in T47D human breast cancer cells before and after prolactinstimulation.

[0081]FIG. 3—Antibody AXI specifically detects tyrosine phophorylatedStat5 by immunoblotting. Immunoblotting of activated Stat5 in COS-7kidney cells transfected with either wild type Sta5 or a tyrosinephosphorylation-defective mutant, Stat5-Y694F.

[0082]FIG. 4—Antibody AX1 specifically detects tyrosine phophorylatedStat5 by immunocytochemistry. Immunocytochemical detection of activatedStat5 in COS-7 kidney cells transfected with either wild type Stat5 or atyrosine phosphorylation-defective mutant, Stat5-Y694F.

[0083]FIG. 5—Immunohistochemical detection of levels of activated Stat5in normal and malignant human breast tissues using antibody AX1.Immunohistochemical detection of activated Stat5 in formalin-fixed,paraffin-embedded normal and malignant human breast tissues.

[0084]FIG. 6—Actuarial curves. Kaplan-Meyer actuarial curves for overallsurvival in breast cancer patients with Stat5 positive versus Stat5negative tumors

[0085]FIG. 7—Survival Function. Kaplan-Meyer actuarial curves forrecurrence of metastatic disease (relapse) in node negative breastcancer patients who had undergone lumpectomy (breast conserving surgery)with Stat5 positive versus Stat5 negative tumors. Note that positiveStat5 activation status is associated with no relapse, indicating thatlumpectomy is a safe procedure for this group of patients.

[0086]FIG. 8—Survival function for Stat5 activation status. Coxregression curves for overall survival in breast cancer patients witheither positive or negative Stat5 activation status of the primarytumor. Note that positive Stat5 activation status predicts improvedresponse to antiestrogen therapy on this overall material ofnode-positive and node-negative breast cancer material.

[0087]FIG. 9—Immunohistochemical analysis of activated Stat5 with Axianti-phosphoTyr-Stat5 antibody and a general antibody to Stat5. Thisfigure documents that at least in post-lactational mouse mammary glands(involution), when Stat5 is known to be turned off, AX1 does not detectactivated Stat5 in cell nuclei (left panel), whereas a general Stat5antibody detect significant levels (right panel). Thus, a general Stat5antibody will not accurately detect levels of active Stat5 even if Stat5is located in the cell nucleus.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0088] Definitions

[0089] In order to provide a clearer understanding of the specificationand claims the following definitions are provided.

[0090] The terms a and an should be understood to refer to at least“one” item, but are not limited to reference to only “one”, unless suchis specifically indicated. Thus, for example, reference to “a” cellrefers to one or more cells.

[0091] Activated Stat5—Activated Stat5 is Stat5 that is detected in thecell nucleus in the tyrosine phosphorylated form. Tyrosinephosphorylated Stat5 has been reported to exist in a dimeric structuralconfiguration that is capable of binding to specific DNA sequences.Activated Stat5 may include Stat5a and Stat5b isoforms, gene products,and/or posttranslationally modified variants thereof, such as forexample proteolytically truncated forms (Wang, Stravopodis et al. 1996;Azam, Lee et al. 1997; Kirken, Malabarba et al. 1997; Meyer, Jucker etal. 1998; Grimley, Dong et al. 1999; Lee, Piazza et al. 1999; Piazza,Valens et al. 2000).

[0092] Stat5 is activated by two distinct, sequential events. Theinitial activation switch of Stat5 involves phosphorylation of atyrosine residue within a conserved C-terminal segment that causesdimerization of Stat5 molecules (Gouilleux, Wakao et al. 1994). A secondcoordinated activation event involves translocation of dimerized Stat5from the cytoplasm into the cell nucleus, which permits Stat5 to bind togene regulatory promoter elements and regulate transcription of specificgenes (Gouilleux, Wakao et al. 1994; Kazansky, Kabotyanski et al. 1999).Because Stat5 not only requires phosphorylation of a specific tyrosineresidue, but also needs to translocate into the cell nucleus in order tofunction as an active DNA-binding transcription factor, amounts oftyrosine phosphorylated Stat5 located within the cell nucleus will moreaccurately reflect the levels of activated Stat5 than overall cellularlevels of tyrosine phosphorylated Stat5. For instance, aberrant tyrosinephosphorylation of Stat5a by hyperactive Src tyrosine kinase has beenshown not to be accompanied by nuclear translocation (Kazansky,Kabotyanski et al. 1999). This observation illustrates that quantitationof tyrosine phosphorylation status alone without assessing nuclearlocalization is not sufficient for accurate determination of levels ofactivated Stat5. Correspondingly, Stat transcription factors may becomedephosphorylated within the cell nucleus and loose the ability to bindto DNA (Haspel and Darnell 1999), making assays that detect nuclearStat5 protein levels alone also not sufficient for determining levels ofactivated Stat5. The definition of activated Stat5 therefore refers toboth nuclear localization and tyrosine phosphorylation.

[0093] Alternatively, antibodies or binding reagents that specificallydetect Stat5 in its active, dimerized (structural) configuration mayalso be used to detect Stat5 that has become phosphorylated andtranslocated to the cell nucleus. For description of suchconformation-specific antibody-derivatives that may not bind directly toa phosphorylation site but still detect the active form of othereffector molecules, including cellular oncogenes Ras and receptors forepidermal growth factor and platelet-deriverd growth factor, see(Panneerselvam, Reitz et al. 1995; Bishayee, Beguinot et al. 1999; Horn,Wittinghofer et al. 1999). Detection of activated, nuclear Stat5 is usedto predict the biological behavior of breast tumors and may also beuseful for diagnosing or monitoring other pathological conditionsinvolving changes in the activation state and expression levels ofStat5, including other forms of cancer.

[0094] Antibody—An “antibody” (interchangeably used in plural form) isan immunoglobulin molecule capable of specific binding to a target, suchas a polypeptide, through at least one antigen recognition site. As usedherein, the term encompasses not only intact antibodies, but alsofragments thereof, mutants thereof, fusion proteins, humanizedantibodies, and any other modified configuration of the immunoglobulinmolecule that comprises an antigen recognition site of the requiredspecificity. An antibody against activated Stat5 is used in the methodsof the invention.

[0095] Antigen—The term “antigen” refers to the target molecule that isspecifically bound by an antibody through its antigen recognition site,such as for example, the activated Stat5 antigen. The antigen may, butneed not be chemically related to the immunogen that stimulatedproduction of the antibody. The antigen may be polyvalent, or it may bea monovalent hapten. Examples of different kinds of antigens that can berecognized by antibodies include polypeptides, polynucleotides, otherantibody molecules, oligosaccharides, complex lipids, drugs, andchemicals. An “immunogen” is an antigen capable of stimulatingproduction of an antibody when injected into a suitable host, usually amammal.

[0096] Compounds may be rendered immunogenic by many techniques known inthe art, including crosslinking or conjugating with a carrier toincrease valency, mixing with a mitogen to increase the immune response,and combining with an adjuvant to enhance presentation.

[0097] Antigen Retrieval Reagent—An “antigen retrieval reagent”facilitates and/or allows binding of immunostaining reagents withepitopes masked by formalin-fixation, by natural binding moieties, or bystructural constraints such as protein folding, or any combination ofthese factors. Antigen retrieval reagents can be used alone or incombination with other physical or physicochemical procedures such asheating or microwave treatment (Boon and Kok 1994; Fresno, Molina et al.1997; Shi, Cote et al. 1997; Brown 1998; McNicol and Richmond 1998;Mighell, Hume et al. 1998; Krenacs, Krenacs et al. 1999).

[0098] Such a reagent expands the range of antibodies useful inimmunohistochemistry as well as reduces the incidence of false negativestaining in over-fixed tissues. Methods of antigen retrieval are knownin the art such as described, for example, in U.S. Pat. Nos. 5,244,787and 5,578,452 and in (Boon and Kok 1994; Fresno, Molina et al. 1997;Shi, Cote et al. 1997; Brown 1998; McNicol and Richmond 1998; Mighell,Hume et al. 1998; Krenacs, Krenacs et al. 1999).

[0099] Any embodiement of the invention may use an antigen-retrievalbuffer of about 1 mM Tris at a pH of about 10.

[0100] Binding Probe—Binding probes are not antibody-based(immunoglobulin based) but still bind with high specificity and affinityto an antigen or antigenic site. For instance, following routinemolecular engineering methods such as those set forth in (Ausubel 1988;Sambrook, Maniatis et al. 1989), those skilled in the art may develop abinding probe containing the Stat5 SH2 (src-homology-2) domain, which isknown to bind with high affinity and specificity to thetyrosine-phosphorylated Stat5 molecule (Liu and Roth 1995; Igarashi,Shigeta et al. 1998; Ariyoshi, Nosaka et al. 2000). Such a binding probecould contain one (monovalent) or several (mulitivalent) Stat5 SH2domains. This binding probe could be engineered or chemically modifiedto contain detection label, which could consist of isotope,fluorescence, enzyme or one or more antigenic sites or “tags” to berecognized by secondary antibodies, which in turn may have similardetection labels attached. Thus a non-antibody based binding probe couldbe generated that is able to specifically detect activated Stat5 that istyrosine phosphorylated and present in the cell nucleus.

[0101] Rational genetic engineering, random mutagenesis, or targetedmolecular evolution in vitro may lead to Stat5-SH2 domains with improvedbinding characteristics (Ariyoshi, Nosaka et al. 2000). Alternatively,peptide-based binding probes may be generated from scratch by selectionof random chemical or genetic libraries for interaction with Stat5 inits activated, dimeric conformation, for example by binding to tyrosinephosphorylated Stat5. General approaches to selection of these types ofbinding probes have been described by numerous authors (Kelly, Liang etal. 1996; Dente, Vetriani et al. 1997; Gram, Schmitz et al. 1997; Doiand Yanagawa 1998; Pellegrini, Liang et al. 1998; Doi and Yanagawa 1999;Gram 1999; Cochrane, Webster et al. 2000; Illgen, Enderle et al. 2000;Messmer, Benham et al. 2000; Zhang, Zhu et al. 2000).

[0102] Cancer Cell—The terms “cancerous cell” or “cancer cell”, usedeither in the singular or plural form, refer to cells that haveundergone a malignant transformation that makes them pathological to thehost organism. Malignant transformation is a single- or multi-stepprocess, which involves in part an alteration in the genetic makeup ofthe cell and/or the gene expression profile. Malignant transformationmay occur either spontaneously, or via an event or combination of eventssuch as drug or chemical treatment, radiation, fusion with other cells,viral infection, or activation or inactivation of particular genes.Malignant transformation may occur in vivo or in vitro, and can ifnecessary be experimentally induced. Malignant cells may be found withinthe well-defined tumor mass or may have metastasized to other physicallocations.

[0103] A feature of cancer cells is the tendency to grow in a mannerthat is uncontrollable by the host, but the pathology associated with aparticular cancer cell may take any form. Primary cancer cells (that is,cells obtained from near the site of malignant transformation) can bereadily distinguished from non-cancerous cells by well-establishedpathology techniques, particularly histological examination. Thedefinition of a cancer cell, as used herein, includes not only a primarycancer cell, but any cell derived from a cancer cell ancestor. Thisincludes metastasized cancer cells, and in vitro cultures and cell linesderived from cancer cells.

[0104] Cell line—A “cell line” or “cell culture” denotes highereukaryotic cells grown or maintained in vitro. It is understood that thedescendants of a cell may not be completely identical (eithermorphologically, genotypically, or phenotypically) to the parent cell.Cells described as “uncultured” are obtained directly from a livingorganism, and have been maintained for a limited amount of time awayfrom the organism: not long enough or under conditions for the cells toundergo substantial replication.

[0105] Clinical Sample—It is understood that a “clinical sample”encompasses a variety of sample types obtained from a subject and usefulin the procedure of the invention, such as for example, a diagnostic ormonitoring test of activated Stat5 levels. The definition encompassessolid tissue samples obtained by surgical removal, a pathology specimen,an archived sample, or a biopsy specimen, tissue cultures or cellsderived therefrom and the progeny thereof, and sections or smearsprepared from any of these sources. Non-limiting examples are samplesobtained from breast tissue, lymph nodes, and breast tumors. Thedefinition also encompasses blood, bone marrow, spinal fluid, and otherliquid samples of biologic origin, and may refer to either the cells orcell fragments suspended therein, or to the liquid medium and itssolutes.

[0106] Control Sample—A control sample is a source of cells or tissuefor comparison purposes. A control sample may include, inter alia,cancer-free breast or mammary tissue or an archived pathology samplecontaining activated Stat5 at various levels for use as positivecontrol, and breast tumor tissue or other tissue showing no Stat5activation as negative control samples.

[0107] Diagnostic Method—A “diagnostic method” may include, but is notlimited to determining the metastatic potential of a tumor ordetermining a patient's prognosis following discovery of a breast tumor.Such diagnostic methods may also be used for determining theeffectiveness of a therapeutic regime used to treat cancer or otherdisease involving the presence of activated Stat5. An example of such atherapeutic treatment is antiestrogen treatment for breast cancer. Theterms “diagnostic method” or “monitoring method” are often usedinterchangeably.

[0108] Differential Result—A “differential” result is generally obtainedfrom an assay in which a comparison is made between the findings of twodifferent assay samples, such as a cancerous cell line and a controlcell line or a cancerous tissue and a control tissue. Thus, for example,“differential levels” of a marker protein, such as Stat5 are observedwhen the level of Stat5 is higher in one tissue sample than another.

[0109] Disease-Free Survival—“Disease-free survival” should beunderstood to mean living free of the disease being monitored. Forexample, if activated Stat5 is used to diagnose or monitor breastcancer, disease-free survival would mean free from detectable breastcancer.

[0110] Metastatic Potential—Metastasis refers to the condition of spreadof cancer from the organ of origin to additional sites in the patients.Therefore, “metastatic potential” as it relates to for example, breastcancer may be considered to be the risk of progression of primarynode-negative cancer from localized disease to disseminated, metastaticdisease.

[0111] Monitoring Method—A “monitoring method” may include, but is notlimited to, following a patient's progress or response to a therapeuticregime after discovery of a breast tumor. Such monitoring methods mayalso be used for determining the effectiveness of a therapeutic regimeused to treat cancer or other diseases involving the presence ofactivated Stat5. An example of such a therapeutic treatment isantiestrogen treatment for breast cancer. Antibodies to activated Stat5are used in monitoring methods of this invention. The terms “diagnosticmethod” or “monitoring method” are often used interchangeably.

[0112] Node Negative Breast Cancer—“Node negative breast cancer” isbreast cancer that is localized to the breast without detectablemetastasis to nearby lymph nodes, thereby indicating a low risk forrecurrence of the cancer after surgery of the primary tumor.

[0113] Pathology—The “pathology” caused by cancer cells within a host isanything that compromises the well-being or normal physiology of thehost. This may involve, but is not limited to abnormal or uncontrollablegrowth of the cancer cell, metastasis, release of cytokines or othersecretory products at an inappropriate level, manifestation of afunction inappropriate for its physiological milieu, interference withthe normal function of neighboring cells, aggravation or suppression ofan inflammatory or immunological response, or the harboring ofundesirable chemical agents or invasive organisms.

[0114] Pharmaceutical Candidate—A “pharmaceutical candidate” or “drugcandidate” is a compound believed to have therapeutic potential, that isto be tested for efficacy against a specific condition, such as forexample a condition having altered activated Stat5 levels (such asbreast cancer). The “screening” of a pharmaceutical candidate refers toconducting an assay that is capable of evaluating the efficacy and/orspecificity of the candidate. In this context, “efficacy” refers to theability of the candidate to affect Stat5 activation levels and/or affectthe cell or organism it is administered to in a beneficial way: forexample, the limitation of the pathology of cancerous cells.

[0115] Prognosis—“Prognosis” as used in this application means thelikelihood of recovery from a disease or the prediction of the probabledevelopment or outcome of a disease. For example, if a sample from apatient with breast cancer is positive for nuclear staining with anantibody to activated Stat5, then the “prognosis” for that patient isbetter than if the sample was negative for activated Stat5 staining.Samples may be scored for activated Stat5 levels on a scale from 0-4 forlevels of antibody staining, where 0 is negative and 1-4 representspositive staining at four semiquantitative steps of increasingintensity. Scores 1-4 can be recoded as positive because each positivescore was associated with significantly reduced risk for relapse andfatal disease when compared to score 0 (negative), but increasingintensity among the positive scores did not provide additional riskreduction. Cox semiparametric proportional hazard analysis can be usedto estimate the prognostic value of activated Stat5. Cutpoint analysishas shown that scores 1-4 differ significantly from 0 in terms ofpredicting overall survival among node-negative breast cancer patients,but did not differ significantly from each other (See FIG. 1).Additional refinement of the quantification procedure may reveal abetter quantitative relationship with the prognosis. The term positiveor negative “Stat5 activation status” of tumors used in this descriptionrefers to scores 0 or scores 1-4, respectively.

[0116] The prognosis of a patient with breast cancer may be based, interalia, at least in part on the metastatic potential of the breast cancerand a relationship to activated Stat5 levels. This description is notmeant to limit the basis for the determination of a patient's prognosis,because those of skill in the art would be aware of other related basesfor determination of the prognosis.

[0117] Relative Amount—The term “relative amount” is used where acomparison is made between a test measurement and a control measurement.Thus, the relative amount of a reagent forming a complex in a reactionis the amount reacting with a test specimen, compared with the amountreacting with a control specimen. The control specimen may be runseparately in the same assay, or it may be part of the same sample (forexample, normal tissue surrounding a malignant area in a tissuesection).

[0118] Scoring—A sample may be “scored” during the diagnosis ormonitoring of breast cancer. In its simplest form, scoring may becategorical negative or positive as judged by visual examination ofsamples by immunohistochemistry. More quantitative scoring involvesjudging the two parameters intensity of staining and the proportion ofstained (“positive”) cells that are sampled. Based on these twoparameters numbers may be assigned that reflect increasing levels ofpositive staining. Allred et al (Allred, Harvey et al. 1998) havedescribed one way of achieving this, which involved scoring bothparameters on a scale from 0 (negative) to 4, and summarizing the scoresof the individual parameters to an overall score. This results in ascale with possible scores of 0, 2, 3, 4, 5, 6, 7 or 8. (Note that ascore of 1 is not possible on Allred's scale). A somewhat simplerscoring method integrates the intensity of nuclear staining and theproportion of cells that display stained nuclei into a combined scalefrom 0 to 4. In practice, the scores 7 and 8 of Allred's scalecorrespond to 4 on the simplified scale. In the same way, scores 5 and 6correspond to 3, scores 3 and 4 to score 2, score 2 corresponds to 1,and, 0 corresponds to 0 on both scales. Either scoring method may beapplied to scoring intensity and proportion of staining of activatedStat5 in the cell nuclei. The terms positive or negative “Stat5activation status” of tumors used in the present description refers tolevels of activated Stat5 that correspond to scores 0 or 1-4 on thesimplified scale, respectively.

[0119] Treatment—“Treatment” of an individual or a cell is any type ofintervention in an attempt to alter the non-treated course of theindividual or cell. For example, treatment of an individual may beundertaken to decrease or limit the pathology caused by a cancerharbored in the individual. Treatment includes but is not limited to a)administration of a composition, such as a pharmaceutical composition,b) administration of a surgical procedure (such as lumpectomy ormodified radical mastectomy), or c) administration of radiation therapy,and may be performed either prophylactically, subsequent to theinitiation of a pathologic event or contact with an etiologic agent.

[0120] Tyrosine-phosphorylated Stat5—“Tyrosine-phosphorylated Stat5”refers to Stat5a phosphorylated on amino acid residue Tyr694 or Stat5bphosphorylated on the homologous amino acid residue Tyr699. Thistyrosine phosphorylation causes the Stat5 molecules to dimerize, and iscritical for the ability of Stat5 to bind to DNA. Tyrosinephosphorylated Stat5 is therefore equated with activated Stat5, althoughonly tyrosine phosphorylated Stat5 that is found in the nucleus maystrictly reflect properly activated Stat5. Tyrosine phosphorylated Stat5that remains located in the cytoplasm is not functionally activated inthe sense that it remains unable to interact with DNA in the cellnucleus and regulate gene transcription.

[0121] Diagnostic Antibodies

[0122] The present invention relates to the use of antibodies againstactivated Stat5, antibody fragments against activated Stat5 and Stat5binding probes. Examples of binding probes that may be used to detectactivated Stat5 that are not antibody or immunoglobulin based includeproteins derived from the phosphotyrosyl-binding SH2 (src-homology-2)domain of Stat5 (Wakao, Gouilleux et al. 1994; Ariyoshi, Nosaka et al.2000), or binding proteins that have been selected for their ability tobind to activated Stat5 by using screening methods for large chemical ormolecular libraries similar to those described in the literature (Kelly,Liang et al. 1996; Dente, Vetriani et al. 1997; Gram, Schmitz et al.1997; Igarashi, Shigeta et al. 1998; Cochrane, Webster et al. 2000). Theprinciples for development of such binding reagents have been describedin detail for other binding probes, and provide means for those skilledin the art to use similar approach to develop probes that bind toactivated Stat5. Further elaboration is now provided for terms relatedto diagnostic antibodies and assays.

[0123] “Fragment” is defined as at least a portion of the variableregions of the immunoglobulin molecule which binds to its target, i.e.the antigen binding region. Some of the constant region of theimmunoglobulin may be included.

[0124] “Antigen-binding region” means that part of the antibody, thefusion protein, or the immunoconjugate of the invention which recognizesthe target or portions thereof

[0125] “Directly” means the use of antibodies coupled to a label. Thespecimen is incubated with the labeled antibody, unbound antibody isremoved by washing, and the specimen may be examined.

[0126] “Indirectly” means incubating the specimen with an unconjugatedantibody, washing and incubating with a marker-conjugated antibody. Themarker may be a fluorochrom, enzyme, isotope, metal, etc. The second or“sandwich” antibody thus reveals the presence of the first.

[0127] The term “Stat5 antibody” as used herein includes whole, intactpolyclonal and monoclonal antibody materials, and chimeric antibodymolecules. The Stat5 antibody described above may include any fragmentsthereof containing the active antigen-binding region of the antibodysuch as Fab, F(ab′)2 and Fv fragments, using techniques well establishedin the art (see, e.g., (Rousseaux, Rousseaux-Prevost et al. 1986)). TheStat5 antibody used in the invention also includes fusion proteins.

[0128] In addition, the present invention encompasses use of antibodiesthat are capable of binding to the same antigenic determinant as theactivated Stat5 antibodies and competing with the antibodies for bindingat that site. These include antibodies having the same antigenicspecificity as the Stat5 antibodies but differing in species origin,isotype, binding affinity or biological functions (e.g., cytotoxicity).For example, class, isotype and other variants of the antibodies of theinvention having the antigen-binding region of the Stat5 antibody can beconstructed using recombinant class-switching and fusion techniquesknown in the art (see, e.g., (Thammana and Scharff 1983; Neuberger,Williams et al. 1984; Spira, Paizi et al. 1996).

[0129] One skilled in the art will appreciate that the invention alsoencompasses the use of immunoglobulin fragments that retain recognitionof the antigen. Such immunoglobulin fragments may include, for example,the Fab′, F(ab′)2, F(v) or Fab fragments, or other antigen recognizingimmunoglobulin fragments. Such immunoglobulin fragments can be prepared,for example, by proteolytic enzyme digestion, using enzymes such aspepsin or papain, reductive alkylation, or recombinant techniques. Thematerials and methods for preparing such immunoglobulin fragments arewell-known to those skilled in the art. See generally, (Matthew andReichardt 1982; Parham, Androlewicz et al. 1982; Lamoyi and Nisonoff1983; Parham 1983).

[0130] An immunoglobulin can be a “chimeric antibody” as that term isrecognized in the art. Also, the immunoglobulin may be a “bifunctional”or “hybrid” antibody, that is, an antibody which may have one arm havinga specificity for one antigenic site, such as a tumor associated antigenwhile the other arm recognizes a different target, for example, a haptenwhich is, or to which is bound, an agent lethal to the antigen-bearingtumor cell. Alternatively, the bifunctional antibody may be one in whicheach arm has specificity for a different epitope of a tumor associatedantigen of the cell to be therapeutically or biologically modified. Inany case, the hybrid antibodies have a dual specificity, preferably withone or more binding sites specific for the hapten of choice or one ormore binding sites specific for a target antigen, for example, anantigen associated with a tumor, an infectious organism, or otherdisease state.

[0131] Biological bifunctional antibodies are described, for example, inEuropean Patent Publication, EPA 0 105 360, to which those skilled inthe art are referred. Such hybrid or bifunctional antibodies may bederived, as noted, either biologically, by cell fusion techniques, orchemically, especially with cross-linking agents or disulfidebridge-forming reagents, and may be comprised of whose antibodies and/orfragments thereof Methods for obtaining such hybrid antibodies aredisclosed, for example, in PCT application WO83/03679 and publishedEuropean Application EPA 0 217 577. Particularly preferred bifunctionalantibodies are those biologically prepared from a “polydome” or“quadroma” or which are synthetically prepared with cross-linking agentssuch as bis-(maleimideo)-methyl ether (“BMME”), or with othercross-linking agents familiar to those skilled in the art.

[0132] In addition the immunoglobin may be a single chain antibody(“SCA”) These may consist of single chain Fv fragments (“scFv”) in whichthe variable light (“V(L)”) and variable heavy (“V(H)”) domains arelinked by a peptide bridge or by disulfide bonds. Also, theimmunoglobulin may consist of single V(H) domains (dAbs) which possessantigen-binding activity. See, e.g., (Ward, Gussow et al. 1989;Glockshuber, Malia et al. 1990; Winter and Milstein 1991).

[0133] As used herein, the term “chimeric antibody” refers to amonoclonal antibody comprising a variable region, i.e. binding region,from one source or species and at least a portion of a constant regionderived from a different source or species, usually prepared byrecombinant DNA techniques. Such murine/human chimeric antibodies arethe product of expressed immunoglobulin genes comprising DNA segmentsencoding murine immunoglobulin variable regions and DNA segmentsencoding human immunoglobulin constant regions. Other forms of chimericantibodies encompassed by the invention are those in which the class orsubclass has been modified or changed from that of the originalantibody. Such “chimeric” antibodies are also referred to as“class-switched antibodies”. Methods for producing chimeric antibodiesinvolve conventional recombinant DNA and gene transfection techniquesnow well known in the art. See, e.g., (Morrison, Johnson et al. 1984).

[0134] In addition, the invention encompasses within its scope use ofimmunoglobulins (as defined above) or immunoglobulin fragments to whichare fused active proteins, for example, an enzyme of the type disclosedin (Neuberger, Williams et al. 1984), PCT application, WO86/01533. Thedisclosure of such products is incorporated herein by reference.

[0135] Furthermore, as noted above, the immunoglobulin (antibody), orfragment thereof, used in the present invention may be polyclonal ormonoclonal in nature. Monoclonal antibodies are the preferredimmunoglobulins, however. The preparation of such polyclonal ormonoclonal antibodies now is well known to those skilled in the art who,of course, are fully capable of producing useful immunoglobulins whichcan be used in the invention. See, e.g., (Kohler and Milstein 1975). Inaddition, monoclonal antibodies which are produced by such hybridomasand which are useful, with the appropriate antigen retrieval procedures,in the practice of the present invention are publicly available fromsources such as Advantex BioReagents LLP, 11950 White Oak Landing,Conroe, Tex. 77385, or Zymed, Inc, 458 Cartlon Court, South SanFrancisco, Calif. 94080.

[0136] Particularly preferred antibodies for use in the presentinvention are monoclonal antibodies which recognize tyrosinephosphorylated, activated Stat5.

[0137] Diagnostic Techniques

[0138] Diagnostic techniques involve the detection and quantitation ofantigens of patients thought to be suffering from carcinoma. Suchantigens can be detected using techniques known in the art such asimmunohistochemistry and immunocytochemistry wherein an antibodyreactive with the antigen is used to detect the presence of the antigenin a tissue sample. These assays, using anti-active Stat5 antibodies cantherefore be used for the detection in tissue of the antigen with whichthe anti-active Stat5 antibodies react and thus predict the metastaticpotential of the tumor. Thus, it is apparent from the foregoing that theStat5 antibodies can be used in most assays involving antigen-antibodyreactions. These assays include, but are not limited to, standardradioimmunoassays (RIA) techniques, both liquid and solid phase, as wellas enzyme-linked immunosorbent assays (ELISA) assays, ELISPOT,immunofluorescence techniques, and other immunocytochemical assays (see,e.g., (Sikora and Smedley 1984)). Preferably, the assay is one which canbe used in situ such as in a biopsy sample to be diagnosed orpathological archived material to directly detect levels of activatedStat5 within the tumor cell nuclei.

[0139] The invention also encompasses diagnostic kits for carrying outthe assays described above. In one embodiment, the diagnostic kitcomprises at least anti-active Stat5 monoclonal antibody, fragmentsthereof, fusion proteins or chimeric antibody of Stat5, or anon-antibody based binding probe specific for activated Stat5, and aconjugate comprising a specific binding partner for the Stat5 antibodyor binding probe and a label capable of producing a detectable signal.The reagents can also include ancillary agents such as buffering agents,antigen retrieval solutions and reagents, and protein stabilizing agents(e.g., polysaccharides). The diagnostic kit can further comprise, wherenecessary, other components of the signal-producing system includingagents for reducing background interference, control reagents or anapparatus or container for conducting the test.

[0140] In another embodiment, the diagnostic kit comprises at least aconjugate of the Stat5 antibodies and a label capable of producing adetectable signal. Ancillary agents as mentioned above can also bepresent.

[0141] Flow Cytometry (FACS Analysis)

[0142] Flow cytometry (FCM), an automated, laser optics-based technique,is used to detect and quantify the levels of antigens or chemicallyreactive substances in isolated cells or cell nuclei in suspension. Theuptake or binding of fluorescent molecules that diagnose changes innuclear DNA content as measured by staining of DNA with propidiumiodide; changes in cell size and granularity as measured by forwardlight scatter and 90 degree side light scatter; down-regulation of DNAsynthesis as measured by decrease in bromodeoxyuridine uptake;alterations in expression of cell surface and intracellular proteins orother antigens as measured by reactivity with specific antibodies; andalterations in plasma membrane composition as measured by the binding offluorescein-conjugated Annexin V protein to the cell surface. Methods inflow cytometry are discussed in (Ormerod 2000).

[0143] Flow cytometric quantitation in breast cancer cells obtained frombiopsies or fine needle aspirates provides relevant information, andallows the characterization and quantitation of breast cancer associatedparameters, like over or under-expression or activation as compared tonormal counterparts, that is suitable for the diagnosis of malignancy orfor residual disease evaluation. It may improve scoring systems forprognostic markers of breast tumors. It allows to find originalprognostic parameters and improves the comparison of different seriesdue to a better definition of positivity (more quantitative).

[0144] Flow cytometry is now widely used for immunophenotyping purposes.It allows, in addition to the determination of the percentage ofpositive cells, to determine the intensity of fluorescent staining, thatcan be converted into antigen density provided that reagents are usedunder saturating concentrations and correct standards of fluorescenceare tested in parallel. The concept of antigen density evaluationappears to improve the efficiency of immune techniques in the monitoringof hemopoietic malignancies (Lavabre-Bertrand, George et al. 1994).

[0145] Therapeutic Regimes for Treating Breast Cancer

[0146] Nearly all patients with breast cancer will have some type ofsurgery. “Lumpectomy” removes only the breast lump and the surroundingarea, or margin, of normal tissue. In most cases, lumpectomy is combinedwith 6 to 7 weeks of radiation therapy following surgery. Thiscombination of lumpectomy and radiation is often referred to as “breastconserving” therapy. Alternatively, in a “modified radical mastectomy”,surgeons remove the entire breast and some of the axillary (underarm)lymph nodes. Modified radical mastectomy is the most common surgery forpatients with breast cancer in whom doctors remove the whole breast.Systemic therapies for breast cancer includes adjuvant antiestrogentreatment and chemotherapy.

[0147] Breast conserving surgery—“Lumpectomy” removes only the breastlump and the surrounding area, or margin, of normal tissue. If cancercells are present at the margin (the edge of the excisional biopsy orlumpectomy specimen), a re-excision can usually be done to remove theremaining cancer. In most cases, lumpectomy is combined with 6 to 7weeks of supplementary radiation therapy following surgery.

[0148] Mastectomy—In a “simple (total) mastectomy” procedure surgeonsremove the entire breast but do not remove any lymph nodes from underthe arm, or muscle tissue from beneath the breast. In a “modifiedradical mastectomy”, surgeons remove the entire breast and some of theaxillary (underarm) lymph nodes. Modified radical mastectomy is the mostcommon surgery for patients with breast cancer in whom doctors removethe whole breast. “Radical mastectomy” removes not only the entirebreast, but axillary lymph nodes, and the chest wall muscles under thebreast as well. The modified radical mastectomy has proved as effectiveas radical mastectomy, which is nowadays rarely performed due todisfiguration and frequent side-effects.

[0149] Lymph node surgery—Regardless of whether a breast cancer patienthas a mastectomy, or a lumpectomy for invasive cancer, the physiciansneed to determine whether the cancer has spread. The regional lymphnodes in the underarm drain lymph from the breast, and are typically thefirst sites of spread. Furthermore, lymph node involvement increases thelikelihood that cancer cells have spread through the blood-stream toother parts of the body.

[0150] While lymph node surgery itself does not improve the chance for acure, this is the only way to accurately determine if the cancer hasspread to the lymph nodes. This usually means removing some or all ofthe lymph nodes in the armpit. Typically 10 to 20 lymph nodes in thearmpit are examined by an operation called “axillary lymph nodedissection”. Although axillary lymph node dissection is a safe procedurewith low rates of serious side effects, efforts are ongoing to developnew ways of detecting the spread of cancer to lymph nodes that are lessinvasive and do not involve a full lymph node dissection. Suchalternative methods include the “sentinel lymph node biopsy” (Orr, Hoehnet al. 1999; Sugg, Ferguson et al. 2000), and new detection methods forbreast cancer cells in bone marrow and blood (Berois, Varangot et al.2000; Braun, Pantel et al. 2000; Fetsch, Cowan et al. 2000; Ikeda,Miyoshi et al. 2000; Kraeft, Sutherland et al. 2000; Zhong, Kaul et al.2000). It is possible that these newer methods in the future may replacelymph node dissection as a means of determining micrometastatic spreadof cancer.

[0151] Sentinel lymph node biopsy—In the sentinel lymph node biopsyprocedure the surgeon finds and removes the ‘sentinel node’—the firstlymph node into which a tumor drains, and therefore the one most likelyto contain cancer cells. In a sentinel lymph node biopsy the surgeoninjects a radioactive substance and/or a blue dye into the area aroundthe tumor. Lymphatic vessels carry these materials into the sentinelnode. The doctor can either see the blue dye or detect the radioactivitywith a geiger counter, and then cuts out the node for examination. Ifthe sentinel node contains cancer, the surgeon will have to perform anaxillary dissection-removal of more lymph nodes in the axilla (armpit).If the sentinel node is cancer-free, the patient and her physicians mayconsider avoiding more lymph node surgery and the potential sideeffects. Although the sentinel node procedure is relatively new and itslong-terin effectiveness is uncertain (Orr, Hoehn et al. 1999; Sugg,Ferguson et al. 2000), it is possible that it will turn out to beequally as effective in determining lymph node spread as the full lymphnode dissection.

[0152] Detection of disseminated cancer cells in blood and bonemarrow—Recent methods for detecting metastatic breast cancer cells inblood (Berois, Varangot et al. 2000; Fetsch, Cowan et al. 2000; Kraeft,Sutherland et al. 2000) or in bone marrow (Braun, Pantel et al. 2000;Ikeda, Miyoshi et al. 2000; Zhong, Kaul et al. 2000) are typically basedon the presence of breast cell-specific cytokeratin markers byimmunodetection or by genetic testing. These new methods may also leadto an alternative approach to lymph node dissection for determiningwhether a breast cancer has spread beyond the local tumor area.

[0153] Radiation therapy—Radiation is used to destroy cancer cells leftbehind in the breast, chest wall, or lymph nodes after surgery.Radiation treatments usually take place 5 days a week over a period of 6to 8 weeks. Side effects most likely to occur include swelling andheaviness in the breast, sunburn-like skin changes in the treated area,and fatigue. Changes to the breast tissue and skin usually go away in 6to 12 months. In some women, the breast becomes smaller and firmer afterradiation therapy. Radiation therapy of axillary (armpit area) lymphnodes can also cause lymphedema. Although generally safe, it is evidentthat radiation therapy comes at a considerable expense and withpotentially serious side-effects. Radiation therapy also involves amajor risk for abnormal fetal development, and cannot be used to treatpregnant women with breast cancer.

[0154] Chemotherapy—Systemic treatment with anti-cancer drugs givenintravenously (injected into a vein) or by mouth. Either way, the drugstravel in the bloodstream and move throughout the entire body. Doctorswho prescribe these drugs (medical oncologists) generally use acombination of medicines proven more effective than a single drug. Forwomen with node-negative breast cancer the most frequently usedchemotherapy options are CMF (cyclophosphamide, methotrexate, andfluorouracil), CAF (cyclophosphamide, doxorubicin), and AC (doxorubicin(Adriamycin) and cyclophosphamide).

[0155] Hormone therapy—Estrogen, a female sex hormone produced by theovaries, promotes growth of some breast cancers. Doctors use severalapproaches to block the effect of estrogen or to lower estrogen levels.The most commonly used antiestrogen drug is tamoxifen, taken daily inpill form, usually for 5 years. Studies show that tamoxifen can reducethe chances of breast cancer coming back after surgery if the breastcancer cells contain receptors for estrogen or progesterone. Tamoxifenmay be used to treat metastatic breast cancer, but also a significantnumber of patients with node-negative cancer receive tamoxifentreatment.

[0156] Anti-estrogen treatment with tamoxifen is, however, associatedwith potentially serious morbidity and side-effects For instance,studies have shown an increase of early-stage endometrial cancer (whichoccurs in the lining of the uterus) among post-menopausal women takingtamoxifen (Cardosi and Fiorica 2000). Another potential side-effect oftamoxifen is deep vein thrombosis, a condition in which blood clots formin the deep blood vessels of the legs and groin. The blood clotssometimes break off and spread to the lungs as a life-threateningcomplication. The risk of stroke is also somewhat increased. Other sideeffects are hot flashes, mood swings, and cataracts (Rennie 1993; Gail,Costantino et al. 1999).

EXAMPLE 1

[0157] Analysis of Levels of Activated Stat5 in Breast Cancer to PredictPrognosis

[0158] It has been established in the present invention that activationof Stat5, a transcription factor which is constitutively activated innormal breast epithelial cells, is gradually lost in lesserdifferentiated human breast tumor cells. Based on analysis of humannormal and malignant breast tissue samples, a positive correlation wasobserved between Stat5 activation and degree of cell differentiation.Cell differentiation, as measured by low histological grade, of a tumoris a known general prognostic factor for cancer. Tumors of higher grade,i.e. lower degree of tumor cell differentiation, is associated with poorprognosis. The prognostic value of Stat5 activation for breast canceroutcome therefore was examined. A simple procedure for antigen retrievalof tyrosine-phosphorylated Stat5 in formalin-fixed cells and tissues wasestablished. The procedure validated the specificity of detection ofantibodies directed to this phosphorylated epitope. The new techniquewas then applied to a material from 553 primary tumors obtained frombreast cancer patients with known disease history and well-characterizedtumors.

[0159] The results showed that Stat5 was activated in approximately 50%of primary breast cancers, and that activated Stat5 was correlated withreduced rate of recurrence and increased overall survival rate. Thiscorrelation was especially strong in patients with node-negativedisease. Activated Stat5 may therefore be the first tumor marker thatwill significantly help to identify a subgroup of low-risk breast cancerpatients with excellent prognosis.

[0160] Materials and Methods

[0161] Cell culture and Transfections used for Validation ofAnti-pTyr-Stat5 Antibody AXI

[0162] T47D human breast cancer cells—T47D cells (American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA)were grown in RPMI 1640 medium (Biofluids, Rockville, Md.) containing10% fetal calf serum (Atlanta Biologicals, Norcross, Ga.), 2 nML-glutamine, and penicillin-streptomycin (50 IU/ml and 50 μg/ml,respectively) at 37° C. with 5% CO₂. Subconfluent cultures of T47D cellswere stimulated with 10 nM human prolactin (Genzyme Diagnostics Inc,cat. no. 80-3910-01) for 30 min at 37 C then fixed in situ with 0.5%paraformaldehyde.

[0163] COS-7 cells for transfection studies—COS-7 cells (American TypeCulture Collection, Fairfax, Va.) were grown in RPMI 1640 medium(Biofluids, Rockville, Md.) containing 10% fetal calf serum (AtlantaBiologicals, Norcross, Ga.), 2 mM L-glutamine, andpenicillin-streptomycin (50 IU/ml and 50 μg/ml, respectively) at 37° C.with 5% CO₂. When COS-7 cells grown in 100 nun dishes reached 60%confluence cotransfections were performed using the FuGENE 6transfection reagent (Roche Molecular Biochemicals, Indianapolis, Ind.).Two micrograms of an expression plasrmid p3PRLR encoding the humanprolactin receptor (Yamashita, Xu et al. 1998) were co-transfected witheither 5 μg of expression plasmid pXM-Stat5a (Liu, Robinson et al. 1995)encoding wild-type Stat5a or with 5 μg of plasmid pXM-Stat5a-Y694F(Yamashita, Xu et al. 1998) encoding a phosphotyrosyl-defective mutantof Stat5a. 24 h after transfection cells were stimulated with 10 nMhuman prolactin (Genzyme Diagnostics, Inc) for 30 min at 37 C.

[0164] Solubilization of Proteins and Immunoblotting—Cells weresolubilized in lysis buffer containing 10 mM Tris-HCl, pH 7.6, 5 mMEDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1mM sodium orthovanadate, 1% Triton X-100, 1 mMphenylmethylsulphonylfluoride, 5 μg/ml aprotinin, 1 μg/ml pepstatin A,and 2 μg/ml leupeptin. Clarified cell lysates were resolved by SDS-PAGEand transferred to polyvinylidene difluoride membranes (Millipore). Amonoclonal antibody AXI specific to Stat5 which is phosphorylated ontyrosine-Y694/699 (Advantex BioReagents LLP, Conroe, Tex.) (1 μg/ml) orpolyclonal rabbit antiserum to Stat5a (Advantex BioReagents, ConroeTex.) (1:3,000) was used as primary antibodies and horseradishperoxidase-conjugated goat antibodies to mouse or rabbit IgG assecondary antibodies in conjunction with enhanced chemiluminesencesubstrate mixture (Amersham Pharmacia Biotech, NJ). Consistent withspecificity of recognition of the key phosphorylation site of Stat5,antibody AX1 recognized prolactin-induced tyrosine phosphorylation ofWT-Stat5a but not of mutant Stat5a-Y694F, which lacks thephosphoacceptor hydroxyl group.

[0165] Immunocytochemistry and Immunohistochemistry of Activated Stat5

[0166] Immunocytochemistry with AX1 in prolactin-stimulated T47D orprolactin-stimulated COS-7 cells expressing WT orphosphotyrosyl-defective Stat5. T47D cells or COS-7 cells transfectedand treated as described above were fixed in 0.5% formaldehyde andallowed to dry on glass slides. Sections of paraffin-embedded,formalin-fixed tissues from normal or malignant human breast weredeparaffinized by 2 washes in xylene for 15 min each, followed byrehydration in graded ethanol. Slides containing deparaffinized tissuesections or fixed cells were microwave-treated for 25 min in apressure-cooker with antigen-retrieval solution (1 mM Tris at pH 10).Other antigen-retrieval solutions also may be used, including phosphatebuffered saline, pH 7.4, or other buffered aqueous solutions of pH 7-10.

[0167] Alternatively, antigen retrieval may be accomplished similar toJones et al (Jones, Welte et al. 1999) as follows. Sections of fixedtissue can be pretreated first with 1 mg/ml of trypsin for 60 min, andsubsequently with 2 N HCl for 60 min at room temperature, followed bytwo 5 min washes in 100 mM borate buffer. Next samples are treated with0.2% of NP-40 for 30 min at room temperature (RT). In addition, betweeneach of these treatments, two 5 min washes in phosphate-buffered saline(PBS) are needed.

[0168] Following the antigen retrieval procedure, endogenous peroxidaseactivity was blocked by incubating slides in 0.3% hydrogen peroxide for10 min at RT, and non-specific binding of IgG was minimized bypreincubation in normal goat serum for 2 h at RT. The primary antibodyAX1 recognizing phosphorylated tyrosine 694/699 of activated Stat5a/b(Advantex BioReagents LLP, Conroe, Tex., Cat no AX1) was diluted in 1%bovine serum albumin (BSA) in PBS and incubated with the samples at afinal concentration of 0.6 mg/ml for 16 h. Antigen-antibody complexeswere detected using biotinylated goat anti-mouse secondary antibody(Biogenex) followed by streptavidin-horseradish-peroxidase complex,using 3,3-(prime) diaminobenzidine (DAB) as chromogen (brown) and Mayerhematoxylin (blue) as counterstain. For controls subtype-specific mouseIgG was used.

[0169] Monoclonal Anti-Phospho-Stat5a/b(Tyr694/699) Antibody AX1

[0170] AX1 is a mouse monoclonal antibody generated against an immunogenconsisting of a phosphopeptide corresponding to the phosphorylatedtyrosine-694 of human Stat5a, KAVDG(phosphoY)VKPQIK, that was conjugatedto keyhole limpet hemocyanin by standard technique (Harlow and Lane1988). This antigen was used for standard immunization of BALB/c mice(Shepherd and Dean 2000).

[0171] Fusion and screening: Lymphocytes from the spleen of mice withthe highest titer were fused with a mouse myeloma cell line. The fusedcells were plated in eight 96 well plates. Wells with cell growth werescreened for IgG production and for specific antibody production bystandard ELISA method and 8 mother clones were selected. Positive wellswere subcloned three times by limited dilution method. Monoclonalantibody production was achieved in serum-free hybridoma cultures andpurification was done by standard Protein A Sepharose affinitychromatography (Harlow and Lane 1988; Shepherd and Dean 2000). Clone AX1specifically recognizes tyrosine phosphorylated isoforms of Stat5, butnot unphosphorylated isoforms. This specificity has been established byimmunoblotting of unphosphorylated and phosphorylated Stat5immunoprecipitated from human breast cancer cells, or otherprolactin-responsive cells, that have either been treated with prolactinfor 30 min to stimulate Stat5 tyrosine phosphorylation, or fromuntreated cells in which Stat5 remains unphosphorylated (See below,Results section, FIG. 2). Furthermore, AX1 does not recognize Stat5mutants in which the Tyr694 residue has been substituted withphenylalanine, which cannot undergo phosphorylation due to lack of thehydroxyl group of the phenyl ring, but otherwise resembles tyrosine (seebelow, Results section, FIGS. 3 and 4).

[0172] Other Anti-Phospho-Tyr Stat5 Antibodies

[0173] Other antibodies to tyrosine phosphorylated Stat5 may also beuseful for detection of activated Stat5 in tissues or cells including amonoclonal anti-Stat5pY antibody from Zymed, Inc (458 Carlton Court,South San Francisco, Calif. 94080; Cat. no. 33-6000), a monoclonalantibody anti-phospho Stat5a/b (Y694/Y699) from Upstate BiotechnologyInc (199 Saranac Ave, Lake Placid, N.Y. 12946; Cat. no. 05-495), and apolyclonal phospho-Tyr-Stat5 antibody from Upstate Biotechnology, Inc(Cat. no. 06-798). Of these, only Zymed's anti-Stat5 is reportedlyuseful in immunohistochernistry (Jones, Welte et al. 1999), although theextensive antigen retrieval procedure used included extended treatmentof tissue sections with proteases. A concern with that antigen retrievalmethod is loss of antigen unless the proteolytic process is verycarefully controlled. Because various batches of proteolytic enzymeswill have different activities, it may be particularly difficult to keepconditions consistent with this extended and elaborate antigen retrievalmethod. No data are currently available about Upstate Biotechnology'santi-phospho-Stat5a/b (pTyr694/699) antibody regarding its usefulness inimmunohistochemistry. There has been no suggestion whatsoever, to useany of the above antibodies in the diagnostic or monitoring methods forcancer as claimed in the current application.

[0174] Obtaining Human Samples

[0175] Human breast cancer cell line T47D was obtained from ATCC(American Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209, USA). Human female breast tissues, both control tissueand that suspected of harboring a carcinoma, were obtained by standardbiopsy and/or surgery methods known to one of skill in the art.

[0176] For example, incisional biopsies, which involve the removal of asmall wedge of tissue from a larger tumor mass, and excisional biopsies,which involve an excision of the entire suspected tumor tissue withlittle or no margin of surrounding normal tissue were used. Aspiration(or fine needle) biopsy, which involves the aspiration of cells andtissue fragments through a needle that has been guided into the suspecttissue, are other examples of suitable tissue extraction methods. Seegenerally (DeVita, Hellman et al. 1982; Calabresi and Schein 1993).

[0177] Breast Cancer Tissue Microarray

[0178] Formalin-fixed and paraffin-embedded tumor specimens were fromthe archives of the Institute for Pathology, University of Basel,Switzerland, and were obtained in the form of a tissue microarraymounted on a glass slide. All tumors were reviewed by a pathologist. Thetissue microarray was constructed as described by (Kononen, Bubendorf etal. 1998). Briefly, a tissue arraying instrument (Beecher Instruments,MD) was used to create holes in a recipient paraffin block and toacquire tissue cores from the donor block by a thin walled needle withan inner diameter of 0.6 mm, held in an X-Y precision guide. Thecylindrical sample was retrieved from the selected region in the donorand extruded directly into the recipient block with defined arraycoordinates. A solid steel wire, closely fit in the tube, was used totransfer the tissue cores into the recipient block. After theconstruction of the recipient block, multiple 5 urn sections were cutwith a microtome using an adhesive-coated tape sectioning system(Instrumedics, NJ). HE-stained sections were used for histologicalverification of tumor tissue on the arrayed samples. The use of tissuemicroarray is not a critical element of the present invention.Alternatively, tumor samples may be used that are mounted byconventional histological technique as larger tissue sections onindividual glass slides.

[0179] Tumor Scoring and Statistical Methods

[0180] Individual breast tumor samples were scored for activated Stat5levels on a scale from 0-4, where 0 is negative and 1-4 representedpositive staining at four semiquantitative steps of increasingintensity. After an initial cutoff analysis, scores 1-4 were recoded aspositive because each positive score was associated with significantlyreduced risk for relapse and fatal disease, especially in node-negativebreast cancer, when compared to score 0 (negative), but increasingintensity among the positive scores did not provide additional riskreduction (FIG. 1). Cox proportional hazard analysis was used toestimate the prognostic value of activated Stat5.

[0181] Cox regression analysis is a semiparametric method for modelingsurvival or time-to-event data in the presence of censored cases (Hosmerand Lemeshow, 1999; Cox, 1972). In contrast to other survival analyses,e.g. Life Tables or Kaplan-Meyer, Cox allows the inclusion of predictorvariables (covariates) in the models. By constructing a Cox regressionmodel, with Stat5 activation status of primary breast tumors as acovariate, it is possible to test hypotheses regarding the correlationof Stat5 activation status of primary breast tumors to time-to-onset ofeither disease relapse (disease-free survival time, or time tometastatic disease), or time to death from the disease (overall survivaltime). Cox regression analysis is also known as Cox proportional hazardanalysis. This method is standard for testing the prognostic value of atumor marker on patient survival time. When used in multivariate mode,the effect of several covariates are tested in parallel so thatindividual covariates that have independent prognostic value can beidentified, i.e. the most useful markers.

[0182] Results

[0183] Validation of technique by immziocytochemistry usinganti-phosphoTyr-Stat5 antibody.

[0184] Phosphospecific antibodies to tyrosine phosphorylated Stat5a/b(pTyr694/pTyr699) were developed and conditions for immunocytochemistryas well as immunohistochemistry of activated, tyrosine phosphorylatedStat5 in isolated cells or in paraffin-embedded tissue were established.

[0185] Immunocytochemistry of formalin-fixed human T47D breast cancercells showed marked nuclear staining with AX1 following 30 min ofprolactin stimulation, consistent with inducible tyrosinephosphorylation and nuclear translocation of Stat5 (FIG. 2A, left andmiddle panels). Unstimulated cells displayed only weak and scatterednuclear staining (FIG. 2A, left panel), and prolactin-stimulated cellstested with control IgG were negative (FIG. 2A, right panel). FIG. 2Bshows the corresponding prolactin-induced tyrosine phosphorylation ofStat5 in human breast cancer cells T47D by immunoblotting. Specificrecognition by AX1 of Stat5 molecules that are phosphorylated on thepositionally conserved tyrosine residue involved in Stat5 dimerizationwas verified by testing of AX1 against the phosphorylation-defectivemutant Y694F. In this mutant the phosphoacceptor hydroxyl group ofTyr694 of Stat5a had been removed through substitution with Phe (FIG.3). For these experiments, COS-7 cells that lacked detectable levels ofendogenous Stat5 were transfected with expression plasmids encodingprolactin receptor and either wild type (WT) Stat5, mutant Stat5(Y694F), or empty control plasmid (Ctrl). Immunoprecipitation of Stat5from transfected cells followed by immunoblotting with AX1 showed thatthis antibody recognized tyrosine phosphorylated Stat5 only inprolactin-stimulated cells that expressed WT-Stat5, and not inunstimulated cells or in prolactin-stimulated cells expressing thephosphotyrosyl-defective Stat5 mutant (FIG. 3, upper panel). Blotting ofreplicate samples with anti-Stat5 antibodies verified that equal levelsof Stat5-WT and Stat5-Y694F were expressed in the transfected cells, andthat no Stat5 was detectable in untransfected COS-7 cells (FIG. 3, lowerpanel). The specificity of reaction of AX1 to Stat5 phosphorylated onthe positionally conserved tyrosine residue also was verified byimmunocytochemistry in COS-7 cells (FIG. 4). As demonstrated by markedand inducible nuclear staining, Stat5 became activated after prolactinstimulation in approximately 30-50% of the COS-7 cells, consistent withexpected transfection efficiencies of these cells (FIG. 4, upperpanels). No inducible nuclear or cytoplasmic staining was observed inCOS-7 cells transfected with either phosphotyrosyl-defective Stat5mutant or with vector control (FIG. 4B, middle and lower panels). Theseinitial experiments therefore demonstrated that antibody AX1specifically recognized the tyrosine phosphorylated form of Stat5, andthat this antibody was useful for immunocytochemical analysis offormalin-fixed cells.

[0186] To test whether AX1 is useful for sections from formalin-fixed,paraffin-embedded tissues, initial screening of normal and malignanthuman breast tissues was performed. This analysis established that AX1was useful for detection of activated Stat5 in sections fromparaffin-embedded, formalin-fixed tissue and established that there weremarked differences in nuclear AX1 staining between human breast tissuespecimens. Specifically, immunohistochemistry of sections ofparaffin-embedded tissues from resting and lactating breast demonstratedthat Stat5 was consistently activated in resting mammary tissue, andeven more markedly in lactating breast tissue (FIG. 5, upper panel).Furthermore, FIG. 5 also shows examples of human primary breastcarcinomas that were either positive or negative for Stat5 activation,including cases of both infiltrating ductal and lobular breast carcinomaspecimens (FIG. 5, middle and lower panels, respectively). Collectively,these experiments proved that antibody AX1 can be used to specificallyrecognize activated, pTyr-Stat5 within the cell nucleus, and is usefulfor immunocytochemical and immunohistochemical analyses in normaltissues and at least breast cancer.

[0187] The levels of activated Stat5 were evaluated in a series of humanprimary breast cancer samples using a phosphorylation-state specificantibody that recognizes tyrosine phosphorylated Stat5 and works informalin-fixed tissue. Taking advantage of tissue microarray technology,the new immunohistochemical procedure was applied simultaneously totissue derived from a total of 611 primary breast tumors from patientswith well-characterized tumors. Long-term follow-up and disease historywas available for 553 of these tumors.

[0188] With the goal of testing whether levels of active Stat5 inprimary breast tumors are a marker of good prognosis, tumor tissues wereanalyzed from 553 separate breast malignancies of variable diseasestage. For these cases, considerable information was available on themajority of tumors, including ER status, PR status, age of patients atdiagnosis, disease stage, and status of axillary lymph nodes (Kononen,Bubendorf et al. 1998). With regard to assessing the role of Stat5 as aprognostic marker in node-negative breast cancer it was of particularinterest to the current study that a relatively large number ofnode-negative tumors were from patients who had not received adjuvantantihormone or chemotherapy.

[0189] After eliminating specimens that were of insufficient quality forconclusive analysis, levels of activated Stat5 were determined in 428 ofthe 553 primary human breast tumor samples. The principal reason forexclusion of as many as 125 cases was not related to a generally poorspecimen quality but rather reflected the technological compromise ofsmall sample sizes available on tissue arrays, where each tumor isrepresented by a circular tissue section of 0.6 mm diameter (Kononen,Bubendorf et al. 1998; Schraml, Kononen et al. 1999). Samples wereprimarily excluded because of lack of sufficient number of tumor cellsin the available section, and was only to a lesser extent due tounsatisfactory or unrepresentative tissue quality.

[0190] Levels of Activated Stat5 are a Marker of Human Breast CancerCell Differentiation

[0191] Levels of activated Stat5 were determined by a semiquantitativescoring method that was based on the combined intensity of nuclear AX1staining and the proportion of tumor cells that showed staining. Sampleswithout nuclear staining were scored as 0 and samples showing nuclearstaining were scored within a range of 1-4 according to the proportionand intensity of nuclear staining of tumor cells. This approachcorresponded to a simplified version of the general immunohistochemicalscoring method described by Allred and colleagues (Allred, Harvey et al.1998). Correlation analysis of this large material established a strongnegative correlation between levels of activated Stat5 and reduced tumorcell differentiation (high grade) as measured by histological grade(Table 1; Spearman correlation coefficient (rho)=−0.346; p<0.001). Aweaker but statistically significant negative correlation also existedbetween levels of activated Stat5 and tumor stage (rho=−0.144; p=0.003).Importantly, there were highly significant positive correlations betweenlevels of activated Stat5 in the primary tumor and both time to relapseof disease (metastasis; rho=0.223; p<0.001) and overall survival time(rho=0.218; p<0.001; see Table 1). These observations provided the firstevidence that Stat5 activation status of primary tumors might hasprognostic significance for breast cancer patients. In contrast, levelsof activated Stat5 in the primary tumor did not correlate with eitherpatient age or number of positive lymph nodes (Table 1). TABLE 1Spearman Rank Correlation Analysis of Levels of Activated Stat5, TumorGrade and Other Selected Parameters Activated Tumor Tumor DiseaseSurvival Stat5 grade stage free interval time Age Tumor gradeCorrelation Coeff. −.346** Sig. (2-tailed) .000 N 428 Tumor stageCorrelation Coeff. −.144** .187** Sig. (2-tailed) .003 .000 N 422 547Disease free interval^(a) Correlation Coeff. .223** −.285** −.232** Sig.(2-tailed) .000 .000 .000 N 428 553 547 Survival time^(a) CorrelationCoeff. .218** −.286** −.192** .934** Sig. (2-tailed) .000 .000 .000 .000N 428 553 547 553 Age Correlation Coeff. −.0 15 −.104* .053 .063 .023Sig. (2-tailed) .753 .015 216 .138 .582 N 428 553 547 553 553 Positivelymph Correlation Coeff. .014 .120** .340** −.333** −.268** −.105* nodesSig. (2-tailed) .780 .006 .000 .000 .000 .017 N 397 519 513 519 519 519

[0192] Positive Stat5 Activation Status Correlates with IntprovedPrognosis of Breast Cancer.

[0193] For subsequent statistical analyses, levels of activated Stat5were recoded as a dichotomous variable that was either negative (score0) or positive (scores 1-4) as described above. The terms negative andpositive Stat5 activation status used in this description refers tolevels of activated Stat5 corresponding to scores 0 and scores 1-4,respectively. Initial analysis of the breast cancer material of 553cases with known disease history revealed that positive Stat5 activationstatus in primary tumors was associated with a moderately reduced riskof disease progression (FIG. 6A). By Kaplan-Meyer analysis (Kaplan1958), the estimated risk of death from breast cancer within 10 years ofsurgical removal of the primary tumor was 25.8% (+/−(SE)4.6%) inpatients with Stat5 positive breast cancer versus 48.9% (+/−7.7%) in theStat5 negative group (p<0.001 by univariate analysis). Proportionalhazard analysis showed that Stat5-negative status was associated with ageneral 2.0-fold increased risk of death from breast cancer (p=0.001). Asimilar protective effect of positive Stat5 status also was observed fordisease-free survival (relapse), with 2.0-fold higher risk forprogression to metastatic disease in patients with Stat5 negative tumors(p<0.001 by univariate proportional hazard analysis; actuarial curvesnot shown). However, when several tumor markers were compared bymultivariate Cox regression analysis, Stat5 status of the primary tumordid not have independent predictive value in this combined material ofboth node-positive and node-negative patients. Instead, node status,tumor stage, histologic grade and progesterone receptor status weremarkers that had independent predictive value on survivorship in thecombined material (FIG. 6A).

[0194] Positive Stat5 Activation Status is a Strong Positive PrognosticMarker for Node-Negative Breast Cancer.

[0195] To test the ability of Stat5 activation status to predict overallsurvival in patients with node-negative disease, the analysis wasconfined to cases with localized tumors. In this patient group (n=272),Stat5 activation status was conclusively determined in 211 cases.Positive Stat5 activation status was associated with significantlyincreased overall survival (FIG. 6B) and disease-free survival (notshown), both by univariate and multivariate analysis (FIG. 6B).Multivariate analysis showed that positive Stat5 activation statuscontinued to be a strong prognostic factor, providing additional andindependent predictive information on overall survival in patients withnode-negative disease, even when other prognostic factors were takeninto account (FIG. 6B). In the material of node-negative tumors, otherthan Stat5 activation status only tumor size had independent prognosticvalue among the currently used prognostic factors, including tumorhistological grade, and hormone receptor status.

[0196] Associated with positive Stat5 status in the primary tumor ofbreast cancer patients with node-negative disease, there was anapproximately 25% survival benefit after 10 years. In this population ofbreast cancer patients, the estimated risk of death from the diseasewithin 10 years of diagnosis based on Kaplan-Meyer analysis wasapproximately eight (8.5) times higher among patients with negativeStat5 activation status in the primary tumor than that of patients withpositive Stat5 activation status (28.3% (+/−7.0%) vs. 3.32% (+/−1.9%);p=0.0001). Proportional hazard analysis estimated the overall relativerisk of death in node-negative patients with negative Stat5 activationstatus of tumors to be 7.7 times the risk associated with Stat5 positivetumors (p=0.001).

[0197] Among node-negative patients in this material a significantfraction had received some form of adjuvant hormone or chemotherapy. Toeliminate the potential bias introduced in case Stat5 status of primarytumors affects the clinical response to adjuvant therapy, and morespecifically assess the pure prognostic value of Stat5 status inpatients with node-negative breast cancer, tumors from patients who hadreceived adjuvant therapy were excluded from the analysis. Survivalanalysis in the remaining population of node-negative patients. (n=114;valid n=86) showed that positive Stat5 activation status continued to bea strong prognostic marker associated with increased overall survival(FIG. 6C) and increased disease-free survival (not shown). Mostimportantly, positive Stat5 activation status in the primary tumor ofnode-negative patients who did not receive adjuvant treatment wasassociated with very high probability of 10 year survival (>97.5%) basedon Kaplan-Meyer estimates (FIG. 5C). Proportional hazard analysis ofthis material of patients with node-negative disease estimated a general11.0-fold higher risk for death when primary tumors were Stat5 negativecompared to that of patients with Stat5 positive tumors (p=0.023).

[0198] Preferably, histological breast tumor markers are prognostic alsoin small tumors. The mean diameter of primary tumors within the entirematerial was 27 mm. To assess whether Stat5 status remained a prognosticfactor also in patients with small, node-negative tumors, the materialof node-negative, adjuvant therapy-free patients was further confined totumors with diameter of 25 nm or less. From a group of 58 cases, nodeaths due to breast cancer were reported over the observation periodamong patients with Stat5 positive status, in contrast to five deathsamong patients with Stat5 negative tumors (FIG. 6D). Despite the limitednumber of cases available for this particular analysis, the observeddifference between the two populations was highly significant (p=0.00⁶by univariate chi-square statistics). Collectively, these resultsclearly suggested that Stat5 activation status will be useful also forpredicting the prognosis of breast cancer patients with small,node-negative tumors.

[0199] The main conclusions from these analyses are:

[0200] 1) Levels of activated Stat5 were strongly correlated withwell-differentiated tumor histology.

[0201] 2) Activated Stat5 status in node-negative tumors was associatedwith a remarkably reduced risk for subsequent disease relapse and deathfrom breast cancer, an observation that was highly significant both byunivariate and multivariate Cox regression analysis.

[0202] The data suggest that Stat5 activation status of individualtumors will significantly help to identify low-risk breast cancerpatients. Stat5 activation status was the most reliable prognosticmolecular/biochemical marker among untreated breast cancer patients withnode negative disease. Knowledge of levels of Stat5 activation status inbreast tumors will distinguish between low and relatively high riskpatients with node-negative breast cancer. Identifying patients withexcellent prognosis will, in general, allow doctors and patients toselect less invasive and less extensive therapeutic approaches (seeExample 2 for more details on therapeutic options). Stat5 activationstatus adds a new level of independent information to current markers ofbreast cancer.

[0203] Activated Stat5 is particularly valuable as a marker of low-riskfor relapse and death in patients without detectable metastases tonearby lymph nodes, so called node-negative breast cancer. This isimportant, because until now, it has been impossible to predict with anyconfidence the risk for relapse (metastatic disease) in this large groupof patients with apparently localized disease.

[0204] In addition to its diagnostic value for breast cancer, activatedStat5 levels should be useful in diagnosing and prognosticating otherforms of cancer where activated Stat5 levels are an indication of tumordifferentiation and metastastic potential. In particular, because of thehigh degree of tissue regulatory similarities between ovaries andmammary glands, including a critical role of Stat5 in maintaining normaldifferentiated function of the ovaries (Teglund, McKay et al. 1998),Stat5 should also be a prognostic marker for localized ovarian cancer.Without being limited by the following mechanism of action, if oneassumes a general differentiative and anti-invasive role of Stat5 invarious tissues, the diagnostic value of activated Stat5 levels shouldalso be applicable to other forms of cancer, including but not limitedto cancer of the ovaries, uterus, large bowel, thyroid, prostate, andskin. In this regard, one of skill in the art should be able to applythe basic methods used for monitoring and diagnosing breast cancer toother cancers of interest.

EXAMPLE 2 Treatment of Breast Cancer

[0205] A sample of breast tissue from a patient with breast cancer orsuspected of having breast cancer is obtained. The sample may be eithera biopsy sample, a pathology sample obtained after a tumor has beenremoved from the breast or an archived sample previously obtained fromthe patient. The sample is analyzed similar to Example 1.

[0206] Based on analysis of levels of activated Stat5 the tumor sample,a treatment regime is determined using acceptable treatment alternativesknown to those skilled in the art. These may include, but are notlimited to, observation, mode of surgery, non-adjuvant therapies such asradiation, and adjuvant therapies such as tamoxifen or cytotoxicchemotherapy.

[0207] The invention has established that a positive Stat5 activationstatus in node-negative breast cancer is associated with a remarkablylow risk for subsequent disease relapse and death from breast cancerwithin 10 years. Specifically, in patients who did not receive anysupplementary antihormone therapy, adjuvant chemotherapy, or neoadjuvantchemotherapy, 10 year estimated survival rate was approximately 97.5%(FIG. 6C). Positive Stat5 activation status in node-negative breastcancer reflects reduced risk of micrometastatic disease. For thoseskilled in the art, knowledge of levels of activated Stat5 in breasttumor biopsies may therefore directly affect the treatment regimechosen.

[0208] It is therefore significant that the present inventiondemonstrates that Stat5 status represents a new and informativeprognostic marker for breast cancer, especially of node-negative breastcancer. Of immediate clinical importance is the exceptionally low riskfor disease progression associated with positive Stat5 status inpatients with node-negative breast tumors. Knowledge of the Stat5activation status in primary breast tumors at the time of diagnosis andsurgical removal may therefore directly influence therapeutic decisionsregarding adjuvant hormone and chemotherapies, as well as supplementaryradiation therapy. In general, the reduced risk of micrometastaticbreast cancer associated with positive Stat5 activation status may beused to favor less invasive and less extensive treatment options, andthus reduce over-treatment of patients with potentially toxic,mutilating or costly procedures and regimes. As will be discussed inmore detail below, this may involve mode of surgery, use ofsupplementary radiation therapy, or use of adjuvant antiestrogen orchemotherapy. This is important because a large number of breast cancerpatients with localized disease currently receive potentially toxic andexpensive supplementary therapies (Thomssen and Janicke 2000).Furthermore, Stat5 status determined in biopsies of primary breasttumors may also help guide surgeons and patients to select betweentissue conserving surgery and radical mastectomy.

[0209] Impact of Stat5 activation status of primary tumors on choice ofsurgery. A series of studies have shown that in breast cancer patientswith node-negative disease (stage I and II), conservation surgery(lumpectomy) is as effective as the modified radical mastectomyprocedure (Reid and Donohue 1992; Noguchi and Miyazaki 1994). Theprincipal advantage of breast conserving therapy is that lumpectomypreserves the appearance of the breast. Nontheless, modified radicalmastectomy remains a widely used procedure, often due to personalpreference of the surgeon and traditions at individual clinics.

[0210] The reduced risk of micrometastatic breast cancer associated withpositive Stat5 activation status could be used to favor less invasivebreast conserving surgery, or lumpectomy, over mastectomy. To examinewhether patients with positive Stat5 activation status could be safelytreated with lumpectomy, occurrence of recurrent disease (relapse) inbreast cancer patients who had undergone lumpectomy were compared withrespect to the Stat5 activation status of their primary tumors (FIG. 7).Among 47 patients with node negative breast cancer with positive Stat5activation status, no failures of lumpectomy treatment were observed. Incontrast, among 51 corresponding patients with negative Stat5 activationstatus, metastatic disease occurred in 7 patients (14%; p=0.003 by logrank analysis) during the observation period. Therefore, we concludethat lumpectomy is a safe procedure for patients with localized breastcancer and positive Stat5 activation status. Thus, Stat5 activationstatus may justify the use of less invasive lumpectomy instead ofmastectomy in breast cancer patients with node-negative disease.

[0211] Impact of Stat5 activation status of primary tumors on use ofsupplementary radiation therapy.

[0212] Although it is generally accepted that lumpectomy is as effectiveas mastectomy on overall survival of breast cancer patients withnode-negative disease (stage I and II), lumpectomy alone is associatedwith a moderately increased risk of local recurrence (Reid and Donohue1992; Schnitt 1998). However, supplementary radiation given followinglumpectomy eliminates this increased risk of local recurrence, and istherefore typically used as a supplement to lumpectomy (Noguchi, Kinneet al. 1996; Taghian and Powell 1999). Nontheless, there is currently asignificant extent of over-treatment with radiation therapy and markershave been sought to better define subpopulations of patients who mightnot need radiation therapy (Morrow, Harris et al. 1995; Marks andProsnitz 1997; Marks and Prosnitz 1997; Schnitt 1998). The reduced riskof micrometastatic breast cancer and disease progression associated withpositive Stat5 activation status therefore could be useful to identify asubpopulation of patients with node-negative disease who might not needsupplementary radiation therapy in addition to lumpectomy.

[0213] Furthermore, there are several subgroups of patients in whomradiotherapy currently is specifically not recommended, which has led tothe recommendation that complete removal of the breast (modified radicalmastectomy) should be used for these groups of patients (1999). Amongthese are pregnant breast cancer patients, due to the harmful effects ofradiation on the developing fetus. Likewise, breast cancer patients withcertain connective tissue diseases that make body tissues especiallysensitive to the side effects of radiation are also currentlyrecommended to undergo mastectomy. Other examples of subgroups ofpatients who are not offered lumpectomy because of the danger ofsupplementary radiation include patients who have already had radiationtherapy to the affected breast or chest, and patients whose initialexcisional biopsy—or, when needed, their reexcision—has not completelyremoved their cancers. The reduced risk of micrometastatic breast cancerassociated with positive Stat5 activation status may define a subgroupof these patients who could be safely treated with lumpectomy withoutthe need for supplementary radiation therapy. Therefore, active Stat5status in primary, node-negative breast cancer patients may identifypatients for which lumpectomy can be used without need for supplementaryradiation therapy.

[0214] Furthermore, also in many cases where a modified radicalmastectomy is selected as the primary treatment, current guidelines makerecommendations as to whether the patient should use supplementaryradiation and/or chemotherapy after surgery (1999). For example, it isrecommended that post-surgery radiation and chemotherapy be used whenthe primary tumor is larger than 5 centimeters, even when the breastcancer is node-negative and the margins are uninvolved with cancer. Thereduced risk of micrometastatic breast cancer associated with positiveStat5 activation status could identify also among these patients withlarge tumors a subgroup with such excellent prognosis that they may notbenefit from supplementary radiation therapy.

[0215] Impact of Stat5 Activation Status of Primary Tumors on Use ofAdjuvant Antiestrogen Therapy

[0216] Stat5 activation status of primary tumors may affect the choiceof antiestrogen treatment in at least two ways. As part of the currentinvention positive Stat5 activation status in primary tumors waspredictive of increased success of antiestrogen treatment by univariateCox regression analysis (odds ratio=1.8, 95% confidence limits 1.0-3.2;p=0.043). Thus, in the absence of other information, positive Stat5activation status of a primary tumor may favor the use to antiestrogentreatment by predicting a moderately improved outcome.

[0217] However, in node-negative breast cancer patients who did notreceive any adjuvant therapy, the exceptionally low risk ofmicrometastatic breast cancer associated with positive Stat5 activationstatus could be useful to eliminate the need for adjuvant antiestrogentherapy in these patients. Thus breast cancer patients with low risk fordistant spread of tumors could be spared from costly and potentiallytoxic adjuvant antiestrogen treatment. This is important because, inaddition to high cost over a typical 5 year treatment period,anti-estrogen treatment with tamoxifen is associated with potentiallyserious morbidity and side-effects. For instance, studies have shown anincrease of early-stage endometrial cancer (which occurs in the liningof the uterus) among post-menopausal women taking tamoxifen (Cardosi andFiorica 2000). Another potential side-effect of tamoxifen is deep veinthrombosis, a condition in which blood clots form in the deep bloodvessels of the legs and groin. The blood clots sometimes break off andspread to the lungs as a life-threatening complication. The risk ofstroke is also somewhat increased. Other side effects are hot flashes,mood swings, and cataracts (Rennie 1993; Gail, Costantino et al. 1999).New tumor markers that will identify patients with very good prognosismay eliminate the need for adjuvant antiestrogen therapy in this groupof patients.

[0218] Impact of Stat5 Activation Status of Primary Tumors on Use ofAdjuvant Chemotherapy

[0219] As for antiestrogen therapy, knowledge of Stat5 activation statusin the primary tumor is useful to significantly help to identifylow-risk breast cancer patients with node negative disease who may bespared from costly and toxic adjuvant chemotherapy. Chemotherapy isfrequently used for node-negative breast cancer and is typicallyadministered over 3-6 months is associated with significant side-effectsand morbidity. Doxorubicin and epirubicin may cause heart damage, butdoctors limit the dose and perform periodic tests to check heartfunction in order to prevent this side effect. Temporary side effectsmight include loss of appetite, nausea and vomiting, mouth sores, hairloss, and changes in the menstrual cycle. Because chemotherapy candamage the blood-producing cells of the bone marrow, a drop in whiteblood cells can raise a patient's risk of infection; a shortage of bloodplatelets can cause bleeding or bruising after minor cuts or injuries;and a decline in red blood cells can lead to fatigue.

[0220] It has been argued that patients can fend off many of these sideeffects. For example, several drugs can prevent or reduce nausea andvomiting. A new group of drugs called growth factors can help bonemarrow recover after chemotherapy and can treat problems resulting fromlow blood counts. However, these drugs, especially growth factors suchas Epogen and Neupogen are very costly, may not prolong survival, andmay come with additional side effects (Del Mastro and Venturini 1998;Meadowcroft, Gilbert et al. 1998; Viens, Genre et al. 1998). Inaddition, patients may also experience permanent complications fromanti-cancer drugs: premature menopause and infertility. The older awomen is when she receives chemotherapy, the more likely she will stopmenstruating or lose her ability to become pregnant. Therefore, if newtumor markers can identify patients with low risk for diseaseprogression, adjuvant chemotherapy with its associated risks andside-effects may not be recommendable for these low-risk patients. Thismay also include neoadjuvant chemoptherapy which sometimes is givenbefore initial surgery.

[0221] Impact of Stat5 Activation Status of Primary Tumors on PatientFollow-Up.

[0222] The excellent prognosis associated with positive Stat5 activationstatus in node-negative breast cancer patients could also affecttreatment by leading to reduced frequency of patient follow-up. Routinesurveillance and follow-up for all patients who have had invasive breastcancer typically includes the following: a history and physical examevery 4-6 months for 2 years, then every 6 months for 3 years, and then,once every year. Women who have had a lumpectomy should undergomammography of the treated breast at 6 months after radiation therapy,and then mammography of both breasts on an annual basis. Becausetamoxifen increases a postmenopausal woman's risk developing cancer ofthe endometrium, postmenopausal patients taking this drug also need tohave an annual pelvic exam.

[0223] If positive Stat5 activation status of primary breast cancer innode-negative patients reduces the need for adjuvant tamoxifen(antiestrogen) therapy, there would be a direct benefit in reduced needfor pelvic examination. More importantly, an excellent prognosis mayallow a general reduction in frequency of follow-up visits to thedoctor's office, a significant benefit in terms of patient time, moneyand quality of life. It will also benefit society by costing less moneyand fewer work days will be lost.

[0224] Impact of Stat5 Activation Status on Treatment Options forRecurrent Tumors.

[0225] A recurrence may be local, meaning that cancer has returned tothe breast, underarm lymph nodes, or nearby tissues, or systemic, whichmeans that cancer has spread to distant organs. For local recurrencewithin the breast tissue in a patient who was first treated withlumpectomy and radiation, current guidelines suggest that a localrecurrence should prompt a mastectomy, and then consideration ofchemotherapy and/or hormonal therapy (1999). The good prognosis and thelow risk of micrometastatic spread of cancer in patients with positiveStat5 activation status in the primary tumor could affect the treatmentof locally recurrent cancer. If monitoring of the recurrent tumorthrough a biopsy shows that the tumor still has positive Stat5activation status, this should signify continued good prognosis, andreexcision using breast conserving surgery might be used instead of thecurrently recommended and more extensive mastectomy.

[0226] Impact of Stat5 Activation Status of Primary Tumors on theDevelopment and Application of New Treatment Approaches to Cancer.

[0227] In addition to affecting the choice and utilization of currentlyavailable breast cancer therapies, knowledge of the Stat5-activationstatus in primary and recurrent breast cancer may be useful forapplication of new breast cancer therapies. Because levels of activatedStat5 in node negative breast cancer are so closely associated withpositive biological behavior of the tumor, regimes to restore activationof Stat5 in breast cancer with negative Stat5 activation status ispredicted to improve outcome by re-establishing a differentiated andless invasive phenotype.

[0228] Therapies to restore Stat5 activation in Stat5 negative breasttumors may include gene therapeutic delivery of Stat5, or genetherapeutic delivery of a Stat5 activating tyrosine kinase, e.g. Jak2,or a combination of both. Alternatively, hyperactive forms of Stat5 maybe used. Examples of hyperactive variants of Stat5 have been describedin the literature, including point-mutants (Ariyoshi, Nosaka et al.2000) and Jak2-Stat5 fusion proteins (Barahmand-Pour, Meinke et al.1998). Such gene therapeutic delivery may involve viral delivery, e.g.adenoviral or adeno-associated viral vectors (Kouraklis 1999; Wu andAtaai 2000), or non-viral gene therapy, e.g. liposome-based delivery ofDNA vectors (Cristiano 1998; Prince 1998). Delivery of viral ornon-viral vectors may be systemic (e.g. intravenous) or local (e.g.intratumoral injection). To achieve more tissue-specific delivery to thetumor tissue, gene delivery may be combined with tissue-specifictargeting strategies, which include but are not limited to tailoring ofviral vectors according to tumor-specific surface markers, use oftumor-targeted inducible vectors that only express the genes of interestwhen a second, tumor-directed drug is used in combination.Alternatively, liposome based non-viral delivery may be targeted totumors by various methods. Such pharmaceutical approaches fortumor-specific gene and drug delivery have been described and reviewedextensively (Huber 1989; Ohno, Levin et al. 1996; Ohno and Meruelo 1996;Berg, Selbo et al. 1999; Patterson and Harris 1999).

[0229] Oher approaches to restore Stat5 activation in breast cancer mayinvolve use of drugs or hormones that specifically upregulate expressionor activation of Stat5, or upregulate expression or activation of one ormore Stat5 activating tyrosine kinases, e.g. Jak2, or inhibit ordown-regulate Stat5 phosphotyrosine phosphatases. Inhibition of Stat5tyrosine phosphatases might be achieved by use of small moleculeinhibitors, antisense-based methods, or introduction ofdominant-negative mutants. Examples of Stat5 activating hormones are thesocalled lactogenic hormones, including human prolactin, human growthhormone, and human placetal lactogen (Bridges 1994; Wartmann, Cella etal. 1996; Handwerger and Freemark 2000). The success of such Stat5targeted therapies may be further monitored in vivo (biopsy afterinitiation of treatment) of patients, or be carried out in vitro ontissue biopsy or cell culture samples, using the method of the presentinvention to detect levels of activated Stat5.

EXAMPLE 3 Method for Screening Compounds

[0230] The pharmaceutical industry is interested in evaluatingpharmaceutically useful compounds which act as growth factor agonists orantagonists. Tens of thousands of compounds per year need to be testedin an entry level or “high flux” screening protocol. Out of thethousands of compounds scrutinized, one or two will show some activityin the entry level assay. These compounds are then chosen for furtherdevelopment and testing. Ideally, a screening protocol would beautomated to handle many samples at once, and would not useradioisotopes or other chemicals that pose safety or disposal problems.An antibody-based approach to evaluating desired or undesired drugregulation of transcription factor activities would provide theseadvantages and offer the added advantage of high selectivity.

[0231] In particular, antibodies that recognize activated Stat5 may beused to for screening drugs in various screening protocols. Generally,two approaches are used. Cell or tissue based approaches use anindicator cell line or tissue that is exposed to the compound to betested. When cells are used it is thought that this approach may quicklyeliminate drugs having solubility or membrane permeability problems.Protein or enzyme-based screens may use purified proteins and canidentify drugs that react with Stat5, or Stat5 tyrosine kinases, orStat5 tyrosine phosphatases, to affect activation state of Stat5.

[0232] For cell or tissue based screening for drugs that modulate (e.g.stimulate, block, inhibit or suppress) Stat5 activation,immunohistochemistry or cytochemistry of Stat5 activation state can beused to measure the effects of individual agents. For example, humanbreast cancer cells, e.g. T47D cells, may be incubated with or withoutprolactin in the presence or absence of drug of interest. Followingactivation with prolactin for 15-30 min, a large proportion ofintracellular Stat5 will normally become tyrosine phosphorylated andtranslocated to the nucleus, while Stat5 will remain unphosphorylated incells not exposed to prolactin (see e.g. FIG. 2). By fixing cells informalin, followed by antigen retrieval and immunocytochemistry foractivated Stat5, the effect of the drug of interest on inducible andbasal Stat5 activation state may be determined.

[0233] Sensitive drug screening methods have been previously proposed inthe art to detect drugs that affect activation state of Stat5 and otherStat-family members based on the ability of activated Stat5 to bind tothe promoter of and drive transcription of Stat-responsive reportergenes that contain the Stat consensus binding site TTNNNNNAA in theirpromoter regions (Lamb et al., U.S. Pat. No. 5,707,803, 1998). Whilethis approach is fairly sensitive, and can be adapted to high throughputformats, the duration of Stat stimulation needed for transcription andtranslation is generally 10-16 h. During this extended time, a series ofevents that are unrelated to Stat activity are sensitive to disruptionby non-specific drugs, including gene transcription, mRNA processing andstability, translation, and protein modification. It is thereforeexpected that a large number of drugs affecting this process will workthrough a non-Stat-dependent mechanism. This will result in many falsepositives, and will require additional control experiments. In addition,this DNA-based approach requires the use of transfection of cells eithertransiently or stably with a reporter gene construct, which limits theuse of this approach to cells that are effectively transfectable andexcludes the use of primary tumor cells or normal cells, which may bemore physiologically and biologically relevant. These disadvantages areovercome by the method of the current invention.

[0234] A second method of studying Stat5 activation that may be usefulfor drug screening is marketed by Cellomics, Inc (635 William Pitt Way,Pittsburgh, Pa. 15238; “Stat5 activation HitKit”; Cat no. K01-0009-1).This method is based on direct detection of Stat5 localized to thenucleus of cells using regular anti-Stat5 antibodies in animmunocytochemical approach. While this method may in many experimentalsettings give an indication of Stat5 activation, this method is lessprecise than the method of the present invention which takes advantageof detecting tyrosine phosphorylated or dimerized Stat5 in the cellnucleus with specific anti-phospho-Stat5alb(pTyr694/699) antibodies.Although there is probably a general correlation between nuclear levelsof Stat5 and degree of Stat5 activation in a given cell, we have foundbased on several experimental models that levels of Stat5 protein incell nuclei frequently do not correspond to the levels of activatedStat5 in the cell nucleus as measured byanti-phospho-Stat5a/b(pTyr694/699) antibodies. Specifically, inpost-lactational mouse mammary glands a huge discrepancy was observed,showing that there can be significant levels of Stat5 detectable in cellnuclei that do not represent active Stat5 as determined byanti-phospho-Stat5alb(pTyr694/699) antibodies in the same samples (FIG.9). In cultured normal rat prostates a similar discrepancy betweennuclear Stat5 protein levels and levels of activated Stat5 as measuredby AX1 antibodies (not shown). Although Cellomic Inc's assay for Stat5activation may be useful in many circumstances, it is evident that theCellomic Inc's methods has the potential to give erroneous results incases where nuclear Stat5 levels do not correspond to Stat5 activation.This disadvantage is overcome by the method of the current invention.

[0235] In contrast to previous methods, the proposed method will be ableto accurately screen for drugs that affect Stat5 activation levels innormal and tumor cells that have not been transfected with artificialDNA. Furthermore, because the proposed screening method assays for Stat5tyrosine phosphorylation and nuclear translocation, which should occurwithin 15-60 min of stimulation or exposure to a candidate drug, anddoes not include a readout that is dependent on proper transcription,mRNA processing, and translation of a gene product, the new methodrepresents a clear improvement to the reporter-gene based assay types.

[0236] An immunohistochemistry-based method that accurately detectslevels of activated Stat5 also has the advantage that it may be usedwith solid tumor explant cultures and organoid cultures, and thereforeallows accurate detection of Stat5-modulating drugs in morephysiologically relevant environmental settings than those used by othermethods. Furthermore, the proposed method will also be applicable toscreening and monitoring the effect of drugs on Stat5 activation statein tissues and cells in research animals and humans in vivo. Samples maybe obtained by biopsy (e.g. fine needle aspiration, section) or bytissue harvesting in the case of research animals and then subjected tothe methods of the invention.

[0237] The proposed method is highly sensitive, because Stat5 activationstate, in principle, may be monitored in a single cell. For practicaluse, more cells may be needed, but good analytic estimates can certainlybe obtained with as little as 20-100 cells.

[0238] The foregoing specification, including the specific embodimentsand examples, is intended to be illustrative of the present inventionand is not to be taken as limiting. Numerous other variations andmodification can be effected without departing from the true spirit andscope of the present invention. All publications, patents and patentapplications cited herein are incorporated by reference in theirentirety into the disclosure.

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[0382]

1 1 1 11 PRT Artificial Sequence Phosphopeptide used in generatingmonoclonal antibodies 1 Lys Ala Val Asp Gly Tyr Val Pro Gln Ile Lys 1 510

What is claimed is:
 1. A diagnostic or monitoring method comprising: a)obtaining a sample of breast tissue from an individual in need ofdiagnosis or monitoring for breast cancer; b) detecting levels ofactivated Stat5 antigen in said sample; c) scoring said sample foractivated Stat5 levels; and d) comparing said scoring to that obtainedfrom a control breast sample to determine the prognosis associated withsaid breast cancer.
 2. The diagnostic or monitoring method of claim 1wherein said breast cancer being diagnosed or monitored is node-negativebreast cancer.
 3. A diagnostic or monitoring method comprising: a)obtaining a sample from an individual in need of diagnosis or monitoringfor breast cancer; b) contacting said sample with an antibody or bindingprobe that detects activated Stat5, c) detecting or measuring the levelof activated Stat5, and d) comparing the level of activated Stat5 tothat obtained from a control breast sample.
 4. The method of claim 1 or3 wherein said detecting or measuring is selected from the group ofmethods consisting of immunoblotting, immunohistochemistry andimmunocytochemistry.
 5. The method of claim 1 or 3 wherein saiddetecting is done by Fluorescence-Activated Cell Sorting (FACS).
 6. Themethod of claim 1 or 3, wherein said sample is contacted or saidactivated Stat5 is detected by an antibody to tyrosine-phosphorylatedStat5.
 7. A diagnostic or monitoring method comprising: a) obtaining asample of breast tissue from an individual in need of diagnosing ormonitoring for breast cancer, b) contacting said sample with an antibodythat detects activated Stat c) detecting or measuring the level ofantibody binding to said sample; and d) comparing the level of antibodybinding in said sample to binding in a control breast sample.
 8. Amethod for screening compounds comprising: a) obtaining compounds to bescreened for use in breast cancer therapy; b) contacting a cell ortissue sample of interest with said compound; and c) determining theeffect of said compound on the level of activated Stat5 in said cell ortissue sample relative to a control sample.
 9. The method of claim 8,wherein said sample is cells or tissue from a breast cancer.
 10. Themethod of claim 7 wherein the effect of said compound is determined bythe binding of an antibody to activated Stat5 to said sample relative tocontrol cells or tissue.
 11. The method of claim 10 wherein saidactivated Stat5 is tyrosine phosphorylated Stat5.
 12. A method fordetermining the effect of antiestrogen treatment comprising: a)obtaining a cell or tissue sample from an individual in need ofantiestrogen treatment; b) measuring the levels of activated Stat5 insaid cell or tissue sample; and c) comparing said levels to that of acontrol breast cancer sample to predict the responsiveness of saidbreast cancer to antiestrogen treatment.
 13. A method for screeningcompounds comprising: a) obtaining compounds to be screened for alteringStat5 activation levels; b) contacting a cell or tissue sample ofinterest with said compound, and c) determining the effect of saidcompound on the level of activated Stat5 in said cell or tissue samplerelative to a control sample.
 14. A kit for determining the level ofactivated Stat5 in a mammalian biological sample wherein said activatedStat5 is an indicator of the prognosis of breast cancer, said kitcomprising: a) an antibody or binding probe to activated Stat5; and b) areagent useful for detecting the extent of interaction between saidantibody or binding probe and activated Stat5; c) a reagent for antigenretrieval; and d) positive and negative control samples.
 15. A methodfor diagnosing a pathological condition or a susceptibility to apathological condition comprising: a) obtaining a sample from anindividual in need of diagnosis for a pathological condition havingaltered levels of activated Stat5; b) determining the amount or presenceof activated Stat5 in said sample; and c) diagnosing said pathologicalcondition or a susceptibility to said pathological condition based onthe presence or altered level of activated Stat5 relative to a controlsample.
 16. The method of any one of claims 1, 3, 4 or 7 wherein theresults are analyzed using univariate or multivariate analysis.
 17. Themethod of any one of claims 1, 3 or 7 wherein said sample is a tissuesection sample.
 18. The method of any one of claims 1, 3, 7 or 12further comprising analyzing the levels of activated Stat5 inconjunction with additional breast cancer markers.
 19. A diagnostic ormonitoring method comprising: a) obtaining a sample of breast tissuefrom an individual in need of diagnosis or monitoring for breast cancer;b) treating said sample in a microwave oven or by other heat-basedmethods of antigen retrieval; c) detecting levels of activated Stat5antigen in said sample; d) scoring said samples for activated Stat5levels; and e) comparing said scoring to that obtained from a controlbreast sample to determine the prognosis associated with said breastcancer.
 20. A diagnostic or monitoring method comprising: a) obtaining asample of breast tissue from an individual in need of diagnosis ormonitoring for breast cancer; b) treating said sample in a microwaveoven or by other heat-based methods of antigen retrieval in anappropriate antigen retrieval solution; c) detecting levels of activatedStat5 antigen in breast cells or breast tissue of said sample; d)scoring said samples for activated Stat5 levels; and e) comparing saidscoring to that obtained from a control breast sample to determine theprognosis associated with said breast cancer.
 21. The method of claim20, wherein said appropriate antigen retrieval solution is an aqueousbuffer with a pH of about 7-10, e.g. phosphate buffered saline, pH 7.4.22. The method of claim 12 further comprising analysis of the activatedStat5 levels between said sample and control by univariate statisticalanalysis.
 23. A method for predicting disease-free survival and overallsurvival in patients with node-negative breast cancer comprising: a)obtaining a sample of breast cancer tissue from an individual withnode-negative breast cancer, b) detecting levels of activated Stat5antigen in breast cancer cells or breast cancer tissue of said sample;c) scoring said samples for activated Stat5 levels; and d) comparingsaid scoring to that obtained from a control breast sample to determinelikelihood of disease-free survival and overall survival associated withsaid breast cancer.
 24. The method of claim 23, wherein said scoringcomprises using a scale of 0 to 4, where 0 is negative (no detectableactivated Stat5 in cell nuclei), and 4 is high intensity staining in themajority of cell nuclei and wherein a score of 1 to 4 (i.e. a positivescore) indicates a better prognosis for disease free and overallsurvival in patients with node-negative breast cancer.
 25. A method forpredicting disease-free survival and overall survival in patients whohave not received adjuvant hormone or chemotherapy comprising: a)obtaining a sample of breast tissue from an individual with breastcancer who has not received adjuvant hormone or chemotherapy; b)detecting levels of activated Stat5 antigen in breast cells or breasttissue of said sample; c) scoring said sample for activated Stat5levels; and d) comparing said scoring to that obtained from a controlbreast sample to determine the likelihood of disease-free survival andoverall survival associated with said breast cancer.
 26. The method ofclaim 25, wherein said scoring comprises using a scale of 0 to 4, where0 is negative (no detectable activated Stat5 in cell nuclei), and 4 ishigh intensity staining in the majority of cell nuclei and wherein ascore of 1 to 4 (i.e. a positive score) indicates a better prognosis fordisease free and overall survival in patients with breast cancer.
 27. Amethod for treating breast cancer comprising: a) obtaining a sample ofbreast tissue from a patient in need of treatment of breast cancer; b)determining the level of activated Stat5 in said breast tissue sample;c) treating said patient with a therapeutic regime known to improve theprognosis for breast cancer; d) repeating steps “a” and “b” e) adjustingthe therapeutic regime known to improve the prognosis for breast cancer;f) repeating steps a-e as frequently as deemed appropriate.
 28. A methodfor screening for metastatic potential of breast tumors comprising: a)obtaining a sample of breast tissue from an individual in need ofscreening for metastatic potential of a breast tumor; b) reacting anantibody to activated Stat5 with tumor tissue from said patient; c)detecting the extent of binding of said antibody to said tissue; and d)correlating the extent of binding of said antibody with metastaticpotential.
 29. The method of claim 28 wherein said tumor is anode-negative breast cancer.
 30. The kit of claim 14 wherein saidantibody is a monoclonal antibody.
 31. The kit of claim 14 wherein saidantibody is a polyclonal antibody.
 32. The kit of claim 14 furthercomprising a second indicator antibody linked to an indicator reagent.33. The kit of claim 32 wherein said indicator reagent is selected fromthe group consisting of fluorescent, calorimetric, immunoperoxidase andisotopic reagents.
 34. A diagnostic or monitoring method comprising: a)obtaining a sample of tissue from an individual in need of diagnosis ormonitoring for cancer; b) detecting levels of activated Stat5 antigen insaid sample, c) scoring said sample for activated Stat5 levels; and d)comparing said scoring to that obtained from a control tissue sample todetermine the prognosis associated with said cancer.
 35. The diagnosticor monitoring method of claim 34 wherein said cancer is selected fromthe group consisting of cancer of the ovary, uterus, thyroid, prostate,skin and large bowel.
 36. A kit for determining the level of activatedStat5 in a mammalian biological sample wherein said activated Stat5 isan indicator of the prognosis of cancer, said kit comprising: a) anantibody or binding probe to activated Stat5; and b) a reagent usefulfor detecting the extent of interaction between said antibody or bindingprobe and activated Stat5; c) a reagent for antigen retrieval; and d)positive and negative control samples.
 37. The kit of claim 36, whereinsaid cancer is a cancer selected from the group consisting of ovariancancer, colorectal cancer, bowel cancer, uterine cancer, endometrialcancer, thyroid gland cancer, prostayic cancer and skin cancer.
 38. Themethod of claim 21 wherein said antigen-retrieval buffer is 1 mM Tris atpH
 10. 39. A monoclonal antibody, wherein said antibody: a) is generatedagainst the phosphopeptide KAVDG(phospho Y)VPQIK); b) specificallyrecognizes tyrosine phosphorylated isoforms of of Stat5, but notunphosphorylated isoforms; c) does not recognize Stat5 mutants in whichthe Tyr694 residue has been substituted with phenylalanine; and d)recognizes phosphorylated Stat5 following an antigen retrieval treatmentthat does not use a protease.
 40. The kit of claim 14, wherein saidantibody is a monoclonal antibody, wherein said antibody: e) isgenerated against the phosphopeptide KAVDG(phospho Y)VPQIK); f)specifically recognizes tyrosine phosphorylated isoforms of of Stat5,but not unphosphorylated isoforms; g) does not recognize Stat5 mutantsin which the Tyr694 residue has been substituted with phenylatanine; andh) recognizes phosphorylated Stat5 following an antigen retrievaltreatment that does not use a protease.